Methods and composition for neutralization of influenza

ABSTRACT

Provided herein are anti-neuraminidase agents useful for neutralization of influenza virus infection, and methods of use and manufacture thereof. In particular, compositions comprising anti-neuraminidase agents (e.g., antibodies) that are cross-reactive with multiple influenza strains are provided, as well as methods of treatment and prevention of influenza infection therewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 62/637,508, filed Mar. 2, 2018, which is incorporated byreference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under U19AI082724,U19AI109946, U19AI057266, awarded by the National Institutes of Health.The government has certain rights in the invention.

FIELD

Provided herein are anti-neuraminidase agents useful for neutralizationof influenza virus, and methods of use and manufacture thereof. Inparticular, compositions comprising anti-neuraminidase agents (e.g.,antibodies) that are cross-reactive with multiple influenza strains areprovided, as well as methods of treatment and prevention of influenzainfection therewith.

BACKGROUND

Influenza is an acute respiratory illness that has caused epidemics andpandemics in the human population for centuries. There are up to 5million cases of influenza virus infection and about 250,000 to 500,000deaths annually around the world (WHO, 2016; herein incorporated byreference in its entirety). The influenza virus has two main surfaceglycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA, the moreabundant protein, mediates binding to sialic acid receptors andsubsequent fusion between the virus and host cell membranes. The lessabundant tetrameric NA protein is essential for cleaving terminal sialicacid residues present on host cell surfaces, allowing the release of thenewly formed viral particles (Matrosovich et al., 2004; Palese andCompans, 1976; herein incorporated by reference in their entireties).Currently, the seasonal influenza virus vaccine is the most widelyavailable method to reduce the annual impact of influenza infection(Nichol, 2008; herein incorporated by reference in its entirety).Antibodies are the primary mediators of protection against influenzainfection (Neu et al., 2016; herein incorporated by reference in itsentirety). Antibodies to HA are typically considered the de factomediators of protection from influenza infection; indeed, inhibition ofHA activity has been the primary measure of influenza vaccine efficacyfor decades. Therefore, most of the current approaches for vaccinedesign focus on inducing an antibody response to influenza virus HA.Influenza vaccine effectiveness can vary widely from season to seasonsuch that protection is always limited and in some years, is quite weak.For example, vaccine effectiveness ranged from only 19% to 48% duringthe past three influenza seasons according to the United States Centersfor Disease Control (Flannery, 2017; herein incorporated by reference inits entirety). Studies have shown that HA antigenic drift (viral genomepoint mutations) is the primary reason for the limited effectiveness ofthe seasonal influenza vaccine (Karron and Collins, 2013; hereinincorporated by reference in its entirety). Due to frequent mutations ofthe HA antigen, especially those located near the receptor bindingdomain, preexisting antibodies often show limited neutralization againstcurrently circulating viruses (Wohlbold and Krammer, 2014; hereinincorporated by reference in its entirety). Although point mutationsalso occur in the NA protein, the rate of antigenic drift around theactive site of NA in the head domain is slower than that for HA amongseasonal influenza A viruses (Abed et al., 2002; Air, 2012; hereinincorporated by reference in its entirety).

SUMMARY

Provided herein are anti-neuraminidase agents useful for neutralizationof influenza virus, and methods of use and manufacture thereof. Inparticular, compositions comprising anti-neuraminidase agents (e.g.,antibodies) that are cross-reactive with multiple influenza strains areprovided, as well as methods of treatment and prevention of influenzainfection therewith.

Provided herein, in part, is the isolation from individuals that havebeen exposed to the influenza virus (e.g., live attenuated virus, fullyinfectious virus, etc.) of antibodies with further selection andcharacterization (e.g., antibodies that bind to NA, human antibodies,monoclonal antibodies, antibody fragments, etc.) that neutralize (e.g.,therapeutically and/or prophylactically) influenza infection (e.g., ofmore than one strains of influenza A virus) and/or inhibit NA activity.In some embodiments, provided herein are epitopes to which theantibodies of the invention bind, and antibodies, antibody fragments,and/or modified antibodies based thereon (e.g., that bind to suchepitopes). Accordingly, in one aspect, provided herein are antibodiesand antigen binding fragments thereof that neutralize influenzainfection (e.g., neutralize infection of one or more than one strain ofinfluenza A virus).

In some embodiments, provided herein are NA-reactive antibodies andantibody fragments that bind to one or more NA types (e.g., N1, N2, N3,N4, N5, N6, N7, N8, N9, N10, and/or N11). In some embodiments, providedherein are NA-reactive antibodies and antibody fragments that cross-bindto heterologous NA proteins (e.g., from human influenza, swineinfluenza, avian influenza, different NA types, etc.).

In some embodiments, provided herein is an isolated antibody, or anantigen binding fragment thereof, that neutralizes infection of an N1strain of influenza (e.g., an H1N1 virus). In another embodiment, anantibody or an antigen-binding fragment thereof also neutralizesinfection of one or more additional NA influenza types (e.g., N2, N3,N4, N5, N6, N7, N8, N9, N10, and/or N11). In some embodiments, anantibody or antibody fragment binds to N309, G249, and/or N273 of N1neuraminidase (e.g., N309 and N273, G249 and N273, etc.).

In some embodiments, provided herein is an isolated antibody, or anantigen binding fragment thereof, that neutralizes infection of an N2strain of influenza (e.g., an H3N2 virus). In another embodiment, anantibody or an antigen-binding fragment thereof also neutralizesinfection of one or more additional NA influenza types (e.g., N1, N3,N4, N5, N6, N7, N8, N9, N10, and/or N11). In some embodiments, anantibody or antibody fragment binds to the conserved enzymatic activesite on the head of N2 neuraminidase.

In certain embodiments, provided herein is an antibody, or antigenbinding fragment thereof, that neutralizes infection of influenza Avirus (e.g., by binding and/or inhibiting NA), wherein the antibody orfragment thereof is expressed by an immortalized B cell clone. In someembodiments, the antibody or fragment thereof is expressed from theimmunoglobulin genes of an isolated B cell.

In some embodiments, provided herein are NA-inhibiting (NI) antibodiesand/or antibody fragments. In some embodiments, antibodies and/orantibody fragments inhibit viral egress from infected cells. In someembodiments, antibodies and/or antibody fragments inhibit release frommucins. In some embodiments, provided herein are non-NI antibodiesand/or antibody fragments.

In another aspect, provided herein are nucleic acids comprising apolynucleotide encoding an antibody or antibody fragment describedherein. In some embodiments, provided herein are vectors comprising anucleic acid molecule or a cell expressing an antibody or an antigenbinding fragment described herein. In some embodiments, provided hereinare cells comprising a vector described herein. In some embodiments,provided herein are isolated or purified immunogenic polypeptidescomprising an epitope that binds to an antibody or antigen bindingfragment described herein.

Also provided herein are pharmaceutical compositions comprising anantibody or an antigen binding fragment described herein, a nucleic acidmolecule described herein, a vector comprising a nucleic acid moleculedescribed herein, a cell expressing an antibody or an antibody fragmentdescribed herein, a cell comprising a vector, or an immunogenicpolypeptide; and a pharmaceutically acceptable diluent or carrier. Insome embodiments, provided herein are pharmaceutical compositionscomprising a first antibody or an antigen binding fragment thereof, anda second antibody, or an antigen binding fragment thereof, wherein thefirst antibody is an antibody described herein, and the second antibodyis any antibody, or antigen binding fragment thereof, that neutralizesinfluenza A or influenza B virus infection.

The use of an antibody or an antigen binding fragment thereof, a nucleicacid, a vector comprising a nucleic acid, a cell expressing a vector, anisolated or purified immunogenic polypeptide comprising an epitope thatbinds to an antibody or antibody fragment described herein, or apharmaceutical composition: (i) in the manufacture of a medicament forthe treatment of influenza A virus infection, (ii) in a vaccine, (iii)in a composition for inducing an immune response, (iv) in diagnosis ofinfluenza A virus infection, or (v) for research purposes, is alsowithin the scope described herein.

In another aspect, provided herein are methods of preventing, treatingor reducing influenza A virus infection or lowering the risk ofinfluenza A virus infection comprising administering to a subject inneed thereof, a therapeutically effective amount of an antibody or anantigen binding antibody fragment of the invention.

Also provided herein are epitopes which are specifically bound by anantibody or an antigen binding fragment described herein, for use (i) intherapy, (ii) in the manufacture of a medicament for treating influenzaA virus infection, (iii) as a vaccine, or (iv) in screening for ligandsable to neutralize influenza A virus infection.

In some embodiments, provided herein are binding agents (e.g.,antibodies or antibody fragments) comprising: (a) a polypeptidecomprising a region having at least 70% sequence identity (e.g., 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, and any ranges with suchendpoints (e.g., 70-100%, 80-100%, 85-99%, 90-99%, etc.)) with apolypeptide of SEQ ID NOs. 2, 18, 34, 50, 66, 82, 98, 114, 130, 146,162, 178, 194, 209, 217, and 225; and (b) a polypeptide comprising aregion having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%,90%, 95%, 98%, 99%, 100%, and any ranges with such endpoints (e.g.,70-100%, 80-100%, 85-99%, 90-99%, etc.)) with a polypeptide of SEQ IDNOs. 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 213,221, and 229; wherein the binding agent exhibits similar influenzaepitope-binding characteristics to an antibody comprising a heavy andlight chain variable regions with 100% sequence identity to those of228-14-035-2D04, 229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04,229-14-036-2C06, 235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02,294-16-009-A-1C06, 294-16-009-A-1D05, 294-16-009-G-1F01,296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02, 229-1F06, and/or229-2D03.

In some embodiments, provided herein are binding agents (e.g.,antibodies or antibody fragments) comprising: (a) a polypeptidecomprising a region having at least 70% sequence identity (e.g., 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, and any ranges with suchendpoints (e.g., 70-100%, 80-100%, 85-99%, 90-99%, etc.)) with apolypeptide of SEQ ID NOs. 2, 18, 34, 50, 66, 82, 98, 114, 130, 146,162, 178, 194, 209, 217, and 225; and (b) a polypeptide comprising aregion having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%,90%, 95%, 98%, 99%, 100%, and any ranges with such endpoints (e.g.,70-100%, 80-100%, 85-99%, 90-99%, etc.)) with a polypeptide of SEQ IDNOs. 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 213,221, and 229; wherein the binding agent exhibits similar influenzaepitope-binding characteristics to an antibody comprising a heavy andlight chain variable regions with 100% sequence identity to those of228-14-035-2D04, 229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04,229-14-036-2C06, 235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02,294-16-009-A-1C06, 294-16-009-A-1D05, 294-16-009-G-1F01,296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02, 229-1F06, and/or229-2D03.

Experiments conducted during development of embodiments herein indicatethe presence of certain amino acids in a neuraminidase enzyme that arerecognized by the antibodies disclosed herein (Table 2). In someembodiments, the amino acids of Table 2 are recognized by binding agentscorresponding to 229-14-036-1D05, 235-15-042-1E06, 294-16-009-A-1C02, or294-16-009-A-1D05, respectively.

TABLE 2 Antibody name Critical amino acid 229-14-036-1D05 N221, G248 andG429 235-15-042-1E06 G248 and G429 294-16-009-A-1C02 N270 and N309294-16-009-A-1D05 N309

In some embodiments, provided herein is a neuraminidase protein (e.g.,recombinant neuraminidase) comprising amino acids of Table 2. In someembodiments, a neuraminidase protein is used to generate or purifytherapeutic antibodies. In some embodiments, provided herein is a virusparticle expressing a recombinant neuraminidase comprising amino acidsof Table 2. In some embodiments, provided herein is a neuraminidaseantigen (e.g., recombinant neuraminidase antigen) comprising the aminoacids of Table 2.

In some embodiments, provided herein are binding agents (e.g.,antibodies or antibody fragments) comprising: (a) a polypeptidecomprising a region having at least 70% sequence identity (e.g., 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, and any ranges with suchendpoints (e.g., 70-100%, 80-100%, 85-99%, 90-99%, etc.)) with apolypeptide encoded by a nucleic acid of SEQ ID NOs. 1, 17, 33, 49, 65,81, 97, 113, 129, 145, 161, 177, and/or 193; and (b) a polypeptidecomprising a region having at least 70% sequence identity (e.g., 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, and any ranges with suchendpoints (e.g., 70-100%, 80-100%, 85-99%, 90-99%, etc.)) with apolypeptide encoded by a nucleic acid of SEQ ID NOs. 9, 25, 41, 57, 73,89, 105, 121, 137, 153, 169, 185, and/or 201; wherein the binding agentexhibits similar influenza epitope-binding characteristics to anantibody comprising a heavy and light chain variable regions with 100%sequence identity to those of 228-14-035-2D04, 229-14-036-1D05,229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06, 235-15-042-1E06,1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06, 294-16-009-A-1D05,294-16-009-G-1F01, 296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02,229-1F06, and/or 229-2D03.

In some embodiments, similar influenza epitope-binding characteristicscomprises: (1) binding to the same epitope, (2) binding to the sameepitope with the same affinity (e.g., as measured by immunofluorescence,ELISA, etc.), binding to the same epitope with less than 10-foldreduction (e.g., 8-fold, 6-fold, 4-fold, 2-fold, etc.) in affinity(e.g., as measured by immunofluorescence, ELISA, etc.).

In some embodiments, the polypeptide of (a) and the polypeptide of (b)comprise first and second polypeptides. In some embodiments, the bindingagent is a monoclonal antibody or monobody. In some embodiments, thebinding agent is an antibody fragment (e.g., Fab, F(ab′)₂, Fab′. scFv,di-scFv, sdAb, etc.). In some embodiments, the polypeptide of (a) andthe polypeptide of (b) are a single polypeptide chain.

In some embodiments, the binding agent comprises a binding affinity foran epitope or epitopes displayed on two or more different virus strains.In some embodiments, the two or more different virus strains areinfluenza strains (e.g., N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and/orN11 influenza stains). In some embodiments, a first influenza strain isan N1 strain (e.g., H1N1). In some embodiments, a first influenza strainis an N2 strain (e.g., H3N2).

In some embodiments, provided herein is a binding agent (e.g., antibody,antibody fragment, etc.) that bind to an epitope of an influenza NAprotein, neutralizes infection of one or more strains of influenza Avirus, and/or inhibits an influenza NA protein, and comprises:

-   -   (i) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 4, a CDR2 of SEQ ID NO: 6 and CDR3 of SEQ ID NO: 8, and a        light chain variable region comprising a CDR1 of SEQ ID NO: 12,        a CDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 16;    -   (ii) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 20, a CDR2 of SEQ ID NO: 22 and CDR3 of SEQ ID NO: 24, and a        light chain variable region comprising a CDR1 of SEQ ID NO: 28,        a CDR2 of SEQ ID NO: 30 and CDR3 of SEQ ID NO: 32;    -   (iii) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 36, a CDR2 of SEQ ID NO: 38 and CDR3 of SEQ ID NO: 40, and a        light chain variable region comprising a CDR1 of SEQ ID NO: 44,        a CDR2 of SEQ ID NO: 46 and CDR3 of SEQ ID NO: 48;    -   (iv) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 52, a CDR2 of SEQ ID NO: 54 and CDR3 of SEQ ID NO: 56, and a        light chain variable region comprising a CDR1 of SEQ ID NO: 60,        a CDR2 of SEQ ID NO: 62 and CDR3 of SEQ ID NO: 64;    -   (v) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 68, a CDR2 of SEQ ID NO: 70 and CDR3 of SEQ ID NO: 72, and a        light chain variable region comprising a CDR1 of SEQ ID NO: 76,        a CDR2 of SEQ ID NO: 78 and CDR3 of SEQ ID NO: 80;    -   (vi) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 84, a CDR2 of SEQ ID NO: 86 and CDR3 of SEQ ID NO: 88, and a        light chain variable region comprising a CDR1 of SEQ ID NO: 92,        a CDR2 of SEQ ID NO: 94 and CDR3 of SEQ ID NO: 96;    -   (vii) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 100, a CDR2 of SEQ ID NO: 102 and CDR3 of SEQ ID NO: 104,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 108, a CDR2 of SEQ ID NO: 110 and CDR3 of SEQ ID NO: 112;    -   (viii) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 116, a CDR2 of SEQ ID NO: 118 and CDR3 of SEQ ID NO: 120,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 124, a CDR2 of SEQ ID NO: 126 and CDR3 of SEQ ID NO: 128;    -   (ix) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 132, a CDR2 of SEQ ID NO: 134 and CDR3 of SEQ ID NO: 136,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 140, a CDR2 of SEQ ID NO: 142 and CDR3 of SEQ ID NO: 144;    -   (x) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 148, a CDR2 of SEQ ID NO: 150 and CDR3 of SEQ ID NO: 152,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 156, a CDR2 of SEQ ID NO: 158 and CDR3 of SEQ ID NO: 160;    -   (xi) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 164, a CDR2 of SEQ ID NO: 166 and CDR3 of SEQ ID NO: 168,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 172, a CDR2 of SEQ ID NO: 174 and CDR3 of SEQ ID NO: 176;    -   (xii) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 180, a CDR2 of SEQ ID NO: 182 and CDR3 of SEQ ID NO: 184,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 188, a CDR2 of SEQ ID NO: 190 and CDR3 of SEQ ID NO: 192;    -   (xiii) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 196, a CDR2 of SEQ ID NO: 198 and CDR3 of SEQ ID NO: 200,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 204, a CDR2 of SEQ ID NO: 206 and CDR3 of SEQ ID NO: 208.    -   (xix) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 210, a CDR2 of SEQ ID NO: 211 and CDR3 of SEQ ID NO: 212,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 214, a CDR2 of SEQ ID NO: 215 and CDR3 of SEQ ID NO: 216;    -   (xx) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 218, a CDR2 of SEQ ID NO: 219 and CDR3 of SEQ ID NO: 220,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 222, a CDR2 of SEQ ID NO: 223 and CDR3 of SEQ ID NO: 224;        and/or    -   (xxi) a heavy chain variable region comprising a CDR1 of SEQ ID        NO: 226, a CDR2 of SEQ ID NO: 227 and CDR3 of SEQ ID NO: 228,        and a light chain variable region comprising a CDR1 of SEQ ID        NO: 230, a CDR2 of SEQ ID NO: 231 and CDR3 of SEQ ID NO: 232.

In some embodiments, provided herein a heavy chain variable regioncomprising:

-   -   (i) a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 6 and CDR3 of        SEQ ID NO: 8, wherein the heavy chain variable region comprises        less than 100% sequence identity (e.g., 99%, 95%, 90%, 85%, 80%,        75%, 70%, or less or ranges therebetween) with SEQ ID NO: 2;    -   (ii) a CDR1 of SEQ ID NO: 20, a CDR2 of SEQ ID NO: 22 and CDR3        of SEQ ID NO: 24, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 18;    -   (iii) a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 38 and CDR3        of SEQ ID NO: 40, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 34;    -   (iv) a CDR1 of SEQ ID NO: 52, a CDR2 of SEQ ID NO: 54 and CDR3        of SEQ ID NO: 56, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 50;    -   (v) a CDR1 of SEQ ID NO: 68, a CDR2 of SEQ ID NO: 70 and CDR3 of        SEQ ID NO: 72, wherein the heavy chain variable region comprises        less than 100% sequence identity (e.g., 99%, 95%, 90%, 85%, 80%,        75%, 70%, or less or ranges therebetween) with SEQ ID NO: 66;    -   (vi) a CDR1 of SEQ ID NO: 84, a CDR2 of SEQ ID NO: 86 and CDR3        of SEQ ID NO: 88, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 82;    -   (vii) a CDR1 of SEQ ID NO: 100, a CDR2 of SEQ ID NO: 102 and        CDR3 of SEQ ID NO: 104, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 98;    -   (viii) a CDR1 of SEQ ID NO: 116, a CDR2 of SEQ ID NO: 118 and        CDR3 of SEQ ID NO: 120, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 114;    -   (ix) a CDR1 of SEQ ID NO: 132, a CDR2 of SEQ ID NO: 134 and CDR3        of SEQ ID NO: 136, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 130;    -   (x) a CDR1 of SEQ ID NO: 148, a CDR2 of SEQ ID NO: 150 and CDR3        of SEQ ID NO: 152, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 146;    -   (xi) a CDR1 of SEQ ID NO: 164, a CDR2 of SEQ ID NO: 166 and CDR3        of SEQ ID NO: 168, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 162;    -   (xii) a CDR1 of SEQ ID NO: 180, a CDR2 of SEQ ID NO: 182 and        CDR3 of SEQ ID NO: 184, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 178;    -   (xiii) a CDR1 of SEQ ID NO: 196, a CDR2 of SEQ ID NO: 198 and        CDR3 of SEQ ID NO: 200, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 194;    -   (xix) a CDR1 of SEQ ID NO: 210, a CDR2 of SEQ ID NO: 211 and        CDR3 of SEQ ID NO: 212, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 209;    -   (xx) a CDR1 of SEQ ID NO: 218, a CDR2 of SEQ ID NO: 219 and CDR3        of SEQ ID NO: 220, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 217; and/or    -   (xxi) a CDR1 of SEQ ID NO: 226, a CDR2 of SEQ ID NO: 227 and        CDR3 of SEQ ID NO: 228, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 225.

In some embodiments, provided herein is a binding agent (e.g., antibody,antibody fragment, etc.) comprising a heavy chain variable region of oneor (i) through (xiii) above.

In some embodiments, provided herein is a light chain variable regioncomprising:

-   -   (i) a CDR1 of SEQ ID NO: 12, a CDR2 of SEQ ID NO: 14 and CDR3 of        SEQ ID NO: 16, wherein the light chain variable region comprises        less than 100% sequence identity (e.g., 99%, 95%, 90%, 85%, 80%,        75%, 70%, or less or ranges therebetween) with SEQ ID NO: 10;    -   (ii) a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 30 and CDR3        of SEQ ID NO: 32, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 26;    -   (iii) a CDR1 of SEQ ID NO: 44, a CDR2 of SEQ ID NO: 46 and CDR3        of SEQ ID NO: 48, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 42;    -   (iv) a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 62 and CDR3        of SEQ ID NO: 64, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 58;    -   (v) a CDR1 of SEQ ID NO: 76, a CDR2 of SEQ ID NO: 78 and CDR3 of        SEQ ID NO: 80, wherein the light chain variable region comprises        less than 100% sequence identity (e.g., 99%, 95%, 90%, 85%, 80%,        75%, 70%, or less or ranges therebetween) with SEQ ID NO: 74;    -   (vi) a CDR1 of SEQ ID NO: 92, a CDR2 of SEQ ID NO: 94 and CDR3        of SEQ ID NO: 96, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 90;    -   (vii) a CDR1 of SEQ ID NO: 108, a CDR2 of SEQ ID NO: 110 and        CDR3 of SEQ ID NO: 112, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 106;    -   (viii) a CDR1 of SEQ ID NO: 124, a CDR2 of SEQ ID NO: 126 and        CDR3 of SEQ ID NO: 128, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 122;    -   (ix) a CDR1 of SEQ ID NO: 140, a CDR2 of SEQ ID NO: 142 and CDR3        of SEQ ID NO: 144, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 138;    -   (x) a CDR1 of SEQ ID NO: 156, a CDR2 of SEQ ID NO: 158 and CDR3        of SEQ ID NO: 160, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 154;    -   (xi) a CDR1 of SEQ ID NO: 172, a CDR2 of SEQ ID NO: 174 and CDR3        of SEQ ID NO: 176, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 170;    -   (xii) a CDR1 of SEQ ID NO: 188, a CDR2 of SEQ ID NO: 190 and        CDR3 of SEQ ID NO: 192, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 186;    -   (xiii) a CDR1 of SEQ ID NO: 204, a CDR2 of SEQ ID NO: 206 and        CDR3 of SEQ ID NO: 208, wherein the light chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 202;    -   (xix) a CDR1 of SEQ ID NO: 214, a CDR2 of SEQ ID NO: 215 and        CDR3 of SEQ ID NO: 216, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 213;    -   (xx) a CDR1 of SEQ ID NO: 222, a CDR2 of SEQ ID NO: 223 and CDR3        of SEQ ID NO: 224, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 221; and/or    -   (xxi) a CDR1 of SEQ ID NO: 230, a CDR2 of SEQ ID NO: 231 and        CDR3 of SEQ ID NO: 232, wherein the heavy chain variable region        comprises less than 100% sequence identity (e.g., 99%, 95%, 90%,        85%, 80%, 75%, 70%, or less or ranges therebetween) with SEQ ID        NO: 229.

In some embodiments, provided herein is a binding agent (e.g., antibody,antibody fragment, etc.) comprising a light chain variable region of oneor (i) through (xiii) above.

In some embodiments, provided herein are methods comprisingadministering a therapeutic dose of a pharmaceutical preparation,composition, and/or formulation described herein (e.g., comprising abinding agents (e.g., antibodies, antibody fragments, etc.) describedherein) to a subject. In some embodiments, the subject is a human ornon-human animal. In some embodiments, the subject is infected withinfluenza (e.g., influenza A). In some embodiments, the subject is atrisk of influenza infection. In some embodiments, the subject isinfected with strain of influenza that expresses a neuraminidaseselected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11. In someembodiments, the binding agent comprises an amino acid sequence that isthe same or is substantially similar (e.g., sequence similarity of 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) or isencoded by a nucleic acid sequence that is the same or is substantiallysimilar (e.g., sequence similarity of 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, 100%, or ranges therebetween) to a sequence described herein (e.g.,SEQ ID NOs: 1-232). In some embodiments, the binding agent is purifiedand/or isolated from a subject that has been infected with influenza. Insome embodiments, the binding agent is the same or is substantiallysimilar (e.g., sequence similarity of 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, 100%, or ranges therebetween) to sequences from a binding agentpurified and/or isolated from a subject that has been infected withinfluenza. In some embodiments, the binding agent is co-administeredwith one or more additional therapeutic agents. In some embodiments, theone or more additional therapeutic agents are selected from the groupconsisting of antivirals, immunologic agents, antibiotics, and agentsfor relieving symptoms of influenza infection.

In some embodiments, provided herein are methods of treating orpreventing an influenza virus infection comprising administering to afirst subject an antibody generated by a second subject infected with aninfluenza virus. In some embodiments, an antibody from the secondsubject is isolated. In some embodiments, an antibody or antibodyfragment comprising the same or similar binding and/or neutralizationcharacteristics (e.g., variable region, CDRs, etc.) to the antibodyisolated from the second subject is administered. In some embodiments,the antibody is a monoclonal antibody. In some embodiments, the antibodyis an antibody fragment. In some embodiments, the antibody is producedby hybridoma, recombinant technology, and/or chemical synthesis. In someembodiments, the antibody administered to the first subject is amodified version of the antibody obtained from second subject.

In some embodiments, provided herein are binding agents (e.g.,antibodies, antibody fragments, etc.) that neutralize infection of oneor more strains of influenza (e.g., influenza A virus). In someembodiments, binding agents bind the same epitope an antibody selectedfrom the group consisting of 228-14-035-2D04, 229-14-036-1D05,229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06, 235-15-042-1E06,1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06, 294-16-009-A-1D05,294-16-009-G-1F01, 296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02,229-1F06, and/or 229-2D03. In some embodiments, the binding agent has anaffinity for the epitope of at least 10⁷ M⁻¹. In some embodiments, thebinding agent comprises variable regions and/or CDRs that are at least70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween)identical to the heavy and light (e.g., lambda or kappa) chains and/orCDRH and CDRL/CDRK of 228-14-035-2D04, 229-14-036-1D05, 229-14-036-1G03,229-14-036-2B04, 229-14-036-2C06, 235-15-042-1E06, 1000-2E06,294-16-009-A-1C02, 294-16-009-A-1C06, 294-16-009-A-1D05,294-16-009-G-1F01, 296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02,229-1F06, and/or 229-2D03.

In some embodiments, provided herein is the use of the antibodies orantibody fragments described herein for the treatment of influenzainfection. In some embodiments, provided herein are the antibodies orantibody fragments described herein for use as a medicament. In someembodiments, provided herein are antibodies or antibody fragments foruse in the treatment of influenza infection. In some embodiments,provided herein is the use of the antibodies or antibody fragmentsdescribed herein for the manufacture of a medicament for the treatmentof influenza infection.

In some embodiments, provided herein is the use of the antibodies,antibody fragments, antigens, and/or epitopes described herein for thediagnosis and/or characterization of an influenza infection. In someembodiments, detection of one or more antigens/epitopes described herein(e.g., using the antibodies/antibody fragments described herein)indicates that a subject or sample is infected with influenza (e.g., aparticular strain or type of influenza). In some embodiments, diagnosticmethods herein find use in directing the treatment of influenzainfection. In some embodiments, provided herein are assays and/ordevices comprising the antibodies, antibody fragments, antigens, and/orepitopes described herein for use in the diagnosis and/orcharacterization of an influenza infection.

In some embodiments, provided herein are quality control reagentscomprising the antibodies, antibody fragments, antigens, and/or epitopesdescribed herein. In some embodiments, provided herein are researchreagents comprising the antibodies, antibody fragments, antigens, and/orepitopes described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Influenza virus infection induces a greater prevalence ofNA-reactive antibodies as compared to vaccination (Panel A) Theproportions of HA-reactive and NA-reactive secreting cells (ASCs) out ofthe total virus-reactive cells were determined by ELISPOT assay.Individuals infected with an H1N1 influenza virus were compared toindividuals infected with an H3N2 influenza virus. Each dot represents asubject (n=6). (Panels B-C) Binding of NA-reactive mAbs to rNA proteinsby ELISA. Represented are ELISA binding curves. The antibody startingconcentration is 10 μg/ml. The assays were performed in duplicate atleast 3 times for each antibody. (Panel B) Binding toA/California/7/2009 (H1N1) rN1 protein or (Panel C) A/Texas/50/2012(H3N2) rN2 protein. (Panels D-E) Proportion of influenza virus-reactivemAbs that bind to HA, NA or other antigens (Panel D) One hundred andtwenty-eight mAbs were isolated from influenza virus infectedindividuals (H1N1 and H3N2). Pie charts show the percentages of mAbsthat bind a given antigen (HA, NA, or other). Graphed on the right arethe percentages of HA- and NA-reactive antibodies per individual. Eachdot represents one individual (n=11). Red indicates patients with no NAB cells detected on first exposure to the pandemic H1N1 strain in 2009(E) Two hundred and fifty-eight mAbs were isolated from influenza virusvaccinated individuals from previously published studies in ourlaboratory (Andrews et al., 2015; Wrammert et al., 2008). As in (PanelD), pie charts show the percentages of mAbs that bind a given antigen(HA, NA, or other) in individuals vaccinated with influenza virussubunit vaccine (seasons 2006-2008 and 2010-2011), influenza virus splitvaccine (2008-2010), or monovalent pandemic H1N1 vaccine (2009-2010).For the panels (Panel A) and (Panel D), the dots indicate patientsinfected with an H3N2 virus.

FIGS. 2A-F. Epitopes on NA are not efficiently presented in currentcommercially available inactivated influenza virus vaccines (A-B) Theproportion of HA and NA-reactive IgG secreting cells (ASCs) in immunizedmice was determined by ELISPOT. Mouse splenocytes were isolated 8 daysafter boost (A) with A/Netherlands/602/2009 (H1N1) virus by intranasalinoculation, or (B) after inactivated A/Switzerland/9715293/2013 (H3N2)virus particle intranasal immunization. Each dot represents one mouse.Pie charts show the average frequency of HA versus NA-reactive B cells.(C-F) HA and NA-reactive mAbs were tested for binding by ELISA to HA, NAand two influenza virus vaccine preparations. Binding avidities (KD)were estimated by Scatchard plot analyses of ELISA data. (C) Binding of35 H1-reactive mAbs to A/California/7/2009 (H1N1) rHA was compared tobinding to influenza virus vaccine Fluarix (2015-2016). Binding of 10H3-reactive mAbs against A/Texas/50/2012 (H3N2) rHA was compared tobinding to vaccine Fluarix (2014-2015), respectively. (E) Binding of 35H1-reactive mAbs to A/California/7/2009 (H1N1) rHA was compared tobinding to the influenza vaccine Fluzone (2016-2017). (D) Binding of 15N1-reactive mAbs to A/California/7/2009 (H1N1) rNA was compared tobinding to influenza virus vaccine Fluarix (2015-2016). Binding of 14N2-reactive mAbs against A/Texas/50/2012 (H3N2) rNA was compared tobinding to vaccine Fluarix (2014-2015), respectively. (F) Binding of 15N1-reactive mAbs to A/California/7/2009 (H1N1) rNA was compared tobinding to the influenza vaccine Fluzone (2016-2017). Data arerepresentative of three independent experiments. Statisticalsignificance was determined using the paired nonparametric Wilcoxontest. The line represents the median. n.s., not significant. *p<0.05;**p<0.001; ***p<0.0001.

FIG. 3. NA-reactive mAbs are broadly cross-reactive. (Panel A) Bindingof NA-reactive mAbs to rNA proteins was measured by ELISA. (Panel A)Representative minimum positive concentrations (μg/ml) from threeindependent experiments are plotted as a heatmap. The different NAs wereclustered by amino acid sequence phylogeny. The top panel showsN2-reactive mAbs binding to a panel of NA proteins. The bottom panelshows N1-reactive mAbs binding to a panel of NA proteins. Pie chartsrepresent the frequency of NA-reactive mAbs binding to historic strains(A/Hong Kong/1/1968 rN2 and A/Brevig Mission/1/1918 rN1). (Panel B)Binding of 32 HA reactive mAbs isolated from infected or vaccinatedsubjects to historical past H3N2 strain (A/Hong Kong/1/1968) rH3 weremeasured by ELISA. Pie charts represent the comparative frequency ofHA-reactive mAbs against A/Hong Kong/1/1968 rH3 protein between theinfected and vaccinated individuals.

FIGS. 4A-C. NA-reactive mAbs exhibit broadly cross-reactiveNA-inhibition and neutralization activity in vitro (A) N2-reactive mAbswere tested for inhibiting NA enzymatic activity via ELLA assays andNA-STAR assays against A/Switzerland/9715293/2013 (H3N2) and A/HongKong/1/1968 (H3N2) viruses. (B) N1-reactive mAbs were tested forinhibiting NA enzymatic activity in ELLA assays and NA-STAR assaysagainst A/California/7/2009 (H1N1) virus and A/Brevig Mission/1/1918(H1N1) rNA protein. (C) NA-reactive mAbs were tested for neutralizationby microneutralization (MN) assay using A/Switzerland/9715293/2013(H3N2) and A/California/7/2009 (H1N1) viruses. Data are represented ashalf-maximum inhibitory concentration (IC50) (μg/ml). (D) Purified N2polyclonal antibodies from infected subjects were tested by MN assayagainst A/Hong Kong/4801/2014 (H3N2) virus. Influenza-non-reactive humanmAb 003-15D3 was used as a negative control in the experiments. Data arerepresented as IC50 (μg/ml). Data are representative of threeindependent experiments.

FIGS. 5A-D. Identification of critical epitopes targeted by NA-reactivemAbs (A) Binding of four N1-reactive mAbs (1000-3B06, 1000-1D05,294-A-1C02 and 294-A-1D05) to A/California/7/2009 (H1N1) NA mutantproteins transiently expressed on the surface of 293T cells.Hyper-immune mouse serum against A/California/7/2009 (H1N1)-X179A viruswas used as a positive control and for examining the expression of NA.Binding to A/California/7/2009 wide type NA is shown in the last barlabeled ‘WT’. Data are represented as mean±SD. Data are representativeof two independent experiments performed in duplicate. (B) Modeling ofN1 was done using PyMOL to show the 4 critical amino acids involved inthe binding of the N1-reactive mAbs (PDB: 3TI6) (Vavricka et al., 2011).(C) Binding of three N2-reactive mAbs (229-1D05, 235-1C02 and 235-1E06)to 12 A/Minnesota/11/2010 (H6N2-PR8 backbone) NA mutant viruses. Dataare represented as mean±SD. Data are representative of two independentexperiments performed in duplicate. (D) Modeling of N2 protein was doneusing PyMOL to show the three critical amino acid involved in thebinding of the N2-reactive mAbs (PDB:4K1J) (Wu et al., 2013).

FIGS. 6A-C. NA-reactive mAbs are protective in a prophylactic setting invivo (A-C) Six week-olds female BALB/c mice (5 per experimentalcondition) were injected intraperitoneally (i.p.) with 5 mg/kg of eachNA-reactive mAb individually or with an irrelevant negative controlhuman mAb 2 h prior to challenge with a lethal dose (10 LD50) of virus.The percentage of initial body weight and survival were plotted for eachantibody and compared to untreated mice. (A) N2-reactive mAbs wereinjected to mice and then infected with 10 LD50 of A/Philippines/2/1982(H3N2-X-79) virus. Percent of initial weight and survival rate areshown. (B) N1-reactive mAbs were injected to mice and then infected with10 LD50 of A/Netherlands/602/2009 virus (pandemic H1N1). Percent ofinitial weight and survival rate are shown. (C) N1-reactive mAbs wereinjected to mice and then infected with 10 LD50 of A/Vietnam/1203/2004(H5N1-PR8 reassortant) avian influenza virus. Percent of initial weightand survival are shown. Data are represented as mean±SD.Influenza-non-reactive human mAb 003-15D3 was used as a negative controlin all experiments.

FIGS. 7A-D. NA-reactive mAbs are protective in a therapeutic setting invivo (A) Binding competition between the N2-reactive mAb 229-1D05 andoseltamivir to A/Texas/50/2012 rNA was measured by bio-layerinterferometry. (B) N2-reactive mAbs were tested for inhibiting NAenzymatic activity via NA-STAR assay against A/Washington/01/2007(oseltamivir-sensitive strain) and A/Texas/12/2007 E119V(oseltamivir-resistant strain) H3N2 viruses. (C-D) Six week-olds femaleBALB/c mice (5 per experimental condition) were infected with a lethaldose (10 LD50) of virus and then administered i.p. with 10 mg/kg ofNA-reactive mAbs or an irrelevant negative control human mAb 48 h afterinfection. The percentage of initial body weight or survival was plottedfor each NA-reactive mAb and compared with untreated mice. (C)N1-reactive mAbs were injected to mice infected with 10LD50 ofA/Netherlands/602/2009 virus (pandemic H1N1). Percent of initial weightand survival are shown. (D) N2-reactive mAbs were administered to miceinfected with A/Philippines/2/1982 (H3N2-X-79) virus. Percent of initialweight and survival rates are shown. Data are represented as mean±SD.Influenza-non-reactive human mAb 003-15D3 was used as a negative controlin all challenge experiments.

FIG. 8. Influenza virus infection induced NA-reactive plasmablasts thatwere VH3-biased. (Panels A-B) The usage of VH immunoglobulin genes by(Panel A) NA-reactive B cells and (Panel B) HA-reactive B cells (PanelC) CDR3 length of NA and HA-reactive mAbs, data are represented asmean±SD. (Panel D) Total mutation number of NA and HA-reactive mAbs,data are represented as mean±SD.

FIGS. 9A-E. Binding competition between 5 N2-reactive mAbs andoseltamivir to A/Texas/50/2012 rNA were measured by bio-layerinterferometry. (A) 229-1F06 (B) 229-1G03 (C) 235-1C02 (D) 235-1E06 (E)229-2C06.

FIG. 10. Heat map of 2014-2015 H3N2-induced NA mAb binding to H3N2strains; x-axis are individual mAbs, y-axis are H3N2 virus strains. Theheat map depicts the mAb clustering based on similarity in viral bindingpatterns using Euclidean distance. The viruses cluster based on theirbinding to the mAbs, they are not clustered based on actual phylogeneticdistance. Each individual box represents the ELISA area under the curvevalue for viral binding, with darker colors being stronger binding.

FIG. 11. Binding curves for 229-1D02 against several H1N1 and H3N2strains.

DEFINITIONS

As used herein, the term “subject” broadly refers to any animal,including but not limited to, human and non-human animals (e.g., dogs,cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As usedherein, the term “patient” typically refers to a subject that is beingtreated for a disease or condition.

As used herein, the term “antibody” refers to a whole antibody moleculeor a fragment thereof (e.g., fragments such as Fab, Fab′, and F(ab′)2),it may be a polyclonal or monoclonal antibody, a chimeric antibody, ahumanized antibody, a human antibody, etc.

A native antibody typically has a tetrameric structure. A tetramertypically comprises two identical pairs of polypeptide chains, each pairhaving one light chain (in certain embodiments, about 25 kDa) and oneheavy chain (in certain embodiments, about 50-70 kDa). In a nativeantibody, a heavy chain comprises a variable region, V_(H), and threeconstant regions, C_(H1), C_(H2), and C_(H3). The V_(H) domain is at theamino-terminus of the heavy chain, and the C_(H3) domain is at thecarboxy-terminus. In a native antibody, a light chain comprises avariable region, V_(L), and a constant region, C_(L). The variableregion of the light chain is at the amino-terminus of the light chain.In a native antibody, the variable regions of each light/heavy chainpair typically form the antigen binding site. The constant regions aretypically responsible for effector function.

In a native antibody, the variable regions typically exhibit the samegeneral structure in which relatively conserved framework regions (FRs)are joined by three hypervariable regions, also called complementaritydetermining regions (CDRs). The CDRs from the two chains of each pairtypically are aligned by the framework regions, which may enable bindingto a specific epitope. From N-terminus to C-terminus, both light andheavy chain variable regions typically comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The CDRs on the heavy chain are referredto as H1, H2, and H3, while the CDRs on the light chain are referred toas L1, L2, and L3. Typically, CDR3 is the greatest source of moleculardiversity within the antigen-binding site. H3, for example, in certaininstances, can be as short as two amino acid residues or greater than26. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat et al. (1991) Sequences ofProteins of Immunological Interest (National Institutes of Health,Publication No. 91-3242, vols. 1-3, Bethesda, Md.); Chothia, C., andLesk, A. M. (1987) J. Mol. Biol. 196:901-917; or Chothia, C. et al.Nature 342:878-883 (1989). In the present application, the term “CDR”refers to a CDR from either the light or heavy chain, unless otherwisespecified.

As used herein, the term “heavy chain” refers to a polypeptidecomprising sufficient heavy chain variable region sequence to conferantigen specificity either alone or in combination with a light chain.

As used herein, the term “light chain” refers to a polypeptidecomprising sufficient light chain variable region sequence to conferantigen specificity either alone or in combination with a heavy chain.

As used herein, when an antibody or other entity “specificallyrecognizes” or “specifically binds” an antigen or epitope, itpreferentially recognizes the antigen in a complex mixture of proteinsand/or macromolecules, and binds the antigen or epitope with affinitywhich is substantially higher than to other entities not displaying theantigen or epitope. In this regard, “affinity which is substantiallyhigher” means affinity that is high enough to enable detection of anantigen or epitope which is distinguished from entities using a desiredassay or measurement apparatus. Typically, it means binding affinityhaving a binding constant (K_(a)) of at least 10⁷ M⁻¹ (e.g., >10⁷ M⁻¹,>10⁸ M⁻¹, >10⁹M⁻¹, >10¹⁰ M⁻¹, >10¹¹ M⁻¹, >10¹² M⁻¹, >10¹³ M⁻¹, etc.). Incertain such embodiments, an antibody is capable of binding differentantigens so long as the different antigens comprise that particularepitope. In certain instances, for example, homologous proteins fromdifferent species may comprise the same epitope.

As used herein, the term “anti-influenza antibody” refers to an antibodywhich specifically recognizes an antigen and/or epitope presented by oneor more strains of influenza virus. A “cross-reactive influenzaantibody” refers to an antibody which specifically recognizes an antigenand/or epitope presented by more than one strain of influenza virus. Forexample, an “N1/N7 cross-reactive influenza antibody” or “N1/N7cross-reactive antibody” specifically recognizes an antigen and/orepitope presented by N1 and N7 strains of influenza.

As used herein, the term “monoclonal antibody” refers to an antibodywhich is a member of a substantially homogeneous population ofantibodies that specifically bind to the same epitope. In certainembodiments, a monoclonal antibody is secreted by a hybridoma. Incertain such embodiments, a hybridoma is produced according to certainmethods known to those skilled in the art. See, e.g., Kohler andMilstein (1975) Nature 256: 495-499; herein incorporated by reference inits entirety. In certain embodiments, a monoclonal antibody is producedusing recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Incertain embodiments, a monoclonal antibody refers to an antibodyfragment isolated from a phage display library. See, e.g., Clackson etal. (1991) Nature 352: 624-628; and Marks et al. (1991) J. Mol. Biol.222: 581-597; herein incorporated by reference in their entireties. Themodifying word “monoclonal” indicates properties of antibodies obtainedfrom a substantially-homogeneous population of antibodies, and does notlimit a method of producing antibodies to a specific method. For variousother monoclonal antibody production techniques, see, e.g., Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.); herein incorporated by referencein its entirety.

As used herein, the term “antibody fragment” refers to a portion of afull-length antibody, including at least a portion antigen bindingregion or a variable region. Antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, Fv, scFv, Fd, diabodies, and otherantibody fragments that retain at least a portion of the variable regionof an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med.9:129-134; herein incorporated by reference in its entirety. In certainembodiments, antibody fragments are produced by enzymatic or chemicalcleavage of intact antibodies (e.g., papain digestion and pepsindigestion of antibody). produced by recombinant DNA techniques, orchemical polypeptide synthesis.

For example, a “Fab” fragment comprises one light chain and the C_(H1)and variable region of one heavy chain. The heavy chain of a Fabmolecule cannot form a disulfide bond with another heavy chain molecule.A “Fab′” fragment comprises one light chain and one heavy chain thatcomprises additional constant region, extending between the C_(H1) andC_(H2) domains. An interchain disulfide bond can be formed between twoheavy chains of a Fab′ fragment to form a “F(ab′)₂” molecule.

An “Fv” fragment comprises the variable regions from both the heavy andlight chains, but lacks the constant regions. A single-chain Fv (scFv)fragment comprises heavy and light chain variable regions connected by aflexible linker to form a single polypeptide chain with anantigen-binding region. Exemplary single chain antibodies are discussedin detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203;herein incorporated by reference in their entireties. In certaininstances, a single variable region (e.g., a heavy chain variable regionor a light chain variable region) may have the ability to recognize andbind antigen.

Other antibody fragments will be understood by skilled artisans.

As used herein, the term “chimeric antibody” refers to an antibody madeup of components from at least two different sources. In certainembodiments, a chimeric antibody comprises a portion of an antibodyderived from a first species fused to another molecule, e.g., a portionof an antibody derived from a second species. In certain suchembodiments, a chimeric antibody comprises a portion of an antibodyderived from a non-human animal fused to a portion of an antibodyderived from a human. In certain such embodiments, a chimeric antibodycomprises all or a portion of a variable region of an antibody derivedfrom a non-human animal fused to a constant region of an antibodyderived from a human.

A “humanized” antibody refers to a non-human antibody that has beenmodified so that it more closely matches (in amino acid sequence) ahuman antibody. A humanized antibody is thus a type of chimericantibody. In certain embodiments, amino acid residues outside of theantigen binding residues of the variable region of the non-humanantibody are modified. In certain embodiments, a humanized antibody isconstructed by replacing all or a portion of a complementaritydetermining region (CDR) of a human antibody with all or a portion of aCDR from another antibody, such as a non-human antibody, having thedesired antigen binding specificity. In certain embodiments, a humanizedantibody comprises variable regions in which all or substantially all ofthe CDRs correspond to CDRs of a non-human antibody and all orsubstantially all of the framework regions (FRs) correspond to FRs of ahuman antibody. In certain such embodiments, a humanized antibodyfurther comprises a constant region (Fc) of a human antibody.

The term “human antibody” refers to a monoclonal antibody that containshuman antibody sequences and does not contain antibody sequences from anon-human animal. In certain embodiments, a human antibody may containsynthetic sequences not found in native antibodies. The term is notlimited by the manner in which the antibodies are made. For example, invarious embodiments, a human antibody may be made in a transgenic mouse,by phage display, by human B-lymphocytes, or by recombinant methods.

As used herein, the term “natural antibody” refers to an antibody inwhich the heavy and light chains of the antibody have been made andpaired by the immune system of a multicellular organism. For example,the antibodies produced by the antibody-producing cells isolated from afirst animal immunized with an antigen are natural antibodies. Naturalantibodies contain naturally-paired heavy and light chains. The term“natural human antibody” refers to an antibody in which the heavy andlight chains of the antibody have been made and paired by the immunesystem of a human subject.

Native human light chains are typically classified as kappa and lambdalight chains. Native human heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. IgG has subclasses,including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM hassubclasses including, but not limited to, IgM1 and IgM2. IgA hassubclasses including, but not limited to, IgA1 and IgA2. Within nativehuman light and heavy chains, the variable and constant regions aretypically joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See, e.g., Fundamental Immunology (1989) Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y.); herein incorporated by reference in itsentirety.

The term “neutralizing antibody” or “antibody that neutralizes” refersto an antibody that reduces at least one activity of a polypeptidecomprising the epitope to which the antibody specifically binds. Incertain embodiments, a neutralizing antibody reduces an activity invitro and/or in vivo. In some embodiments, by neutralizing thepolypeptide comprising the epitope, the neutralizing antibody inhibitsthe capacity of the organism (or virus) displaying the epitope. Forexample, an “influenza neutralizing antibody” reduces the capacity ofone or more strains of influenza to infect a subject.

The term “antigen-binding site” refers to a portion of an antibodycapable of specifically binding an antigen. In certain embodiments, anantigen-binding site is provided by one or more antibody variableregions.

The term “epitope” refers to any polypeptide determinant capable ofspecifically binding to an immunoglobulin or a T-cell receptor. Incertain embodiments, an epitope is a region of an antigen that isspecifically bound by an antibody. In certain embodiments, an epitopemay include chemically active surface groupings of molecules such asamino acids, sugar side chains, phosphoryl, or sulfonyl groups. Incertain embodiments, an epitope may have specific three-dimensionalstructural characteristics (e.g., a “conformational” epitope) and/orspecific charge characteristics.

An epitope is defined as “the same” as another epitope if a particularantibody specifically binds to both epitopes. In certain embodiments,polypeptides having different primary amino acid sequences may compriseepitopes that are the same. In certain embodiments, epitopes that arethe same may have different primary amino acid sequences. Differentantibodies are said to bind to the same epitope if they compete forspecific binding to that epitope.

As used herein, the term “artificial” refers to compositions and systemsthat are designed or prepared by man, and are not naturally occurring.For example, an artificial polypeptide (e.g., antibody or antibodyfragment) or nucleic acid is one comprising a non-natural sequence(e.g., a polypeptide without 100% identity with a naturally-occurringprotein or a fragment thereof).

The term “amino acid” refers to natural amino acids, unnatural aminoacids, and amino acid analogs, all in their D and L stereoisomers,unless otherwise indicated, if their structures allow suchstereoisomeric forms.

Natural amino acids include alanine (Ala or A), arginine (Arg or R),asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C),glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), Lysine(Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline(Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp orW), tyrosine (Tyr or Y) and valine (Val or V).

Unnatural amino acids include, but are not limited to,azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, naphthylalanine (“naph”), aminopropionic acid,2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid,2-aminopimelic acid, tertiary-butylglycine (“tBuG”),2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,homoproline (“hPro” or “homoP”), hydroxylysine, allo-hydroxylysine,3-hydroxyproline (“3Hyp”), 4-hydroxyproline (“4Hyp”), isodesmosine,allo-isoleucine, N-methylalanine (“MeAla” or “Nime”), N-alkylglycine(“NAG”) including N-methylglycine, N-methylisoleucine,N-alkylpentylglycine (“NAPG”) including N-methylpentylglycine.N-methylvaline, naphthylalanine, norvaline (“Norval”), norleucine(“Norleu”), octylglycine (“Octan”), ornithine (“Orn”), pentylglycine(“pG” or “PGly”), pipecolic acid, thioproline (“ThioP” or “tPro”),homoLysine (“hLys”), and homoArginine (“hArg”).

The term “amino acid analog” refers to a natural or unnatural amino acidwhere one or more of the C-terminal carboxy group, the N-terminal aminogroup and side-chain functional group has been chemically blocked,reversibly or irreversibly, or otherwise modified to another functionalgroup. For example, aspartic acid-(beta-methyl ester) is an amino acidanalog of aspartic acid; N-ethylglycine is an amino acid analog ofglycine; or alanine carboxamide is an amino acid analog of alanine.Other amino acid analogs include methionine sulfoxide, methioninesulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteinesulfoxide and S-(carboxymethyl)-cysteine sulfone.

As used herein, the term “artificial polypeptide”, “artificialantibody”, or “artificial binding agent”, consistent with the definitionof “artificial” above, refers to a polypeptide, antibody, or bindingagent having a distinct amino acid sequence or chemical makeup fromthose found in natural polypeptides, antibodies, and binding agents. Anartificial polypeptide or antibody is not a subsequence of a naturallyoccurring protein, either the wild-type (i.e., most abundant) or mutantversions thereof Δn “artificial polypeptide”, “artificial antibody”, or“artificial binding agent”, as used herein, may be produced orsynthesized by any suitable method (e.g., recombinant expression,chemical synthesis, enzymatic synthesis, purification from whole animal,etc.).

As used herein, a “conservative” amino acid substitution refers to thesubstitution of an amino acid in a peptide or polypeptide with anotheramino acid having similar chemical properties, such as size or charge.For purposes of the present disclosure, each of the following eightgroups contains amino acids that are conservative substitutions for oneanother:

1) Alanine (A) and Glycine (G);

2) Aspartic acid (D) and Glutamic acid (E);

3) Asparagine (N) and Glutamine (Q);

4) Arginine (R) and Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V);

6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W);

7) Serine (S) and Threonine (T); and

8) Cysteine (C) and Methionine (M).

Naturally occurring residues may be divided into classes based on commonside chain properties, for example: polar positive (histidine (H),lysine (K), and arginine (R)); polar negative (aspartic acid (D),glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine(N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V),leucine (L), isoleucine (I), methionine (M)); non-polar aromatic(phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine;and cysteine. As used herein, a “semi-conservative” amino acidsubstitution refers to the substitution of an amino acid in a peptide orpolypeptide with another amino acid within the same class.

In some embodiments, unless otherwise specified, a conservative orsemi-conservative amino acid substitution may also encompassnon-naturally occurring amino acid residues that have similar chemicalproperties to the natural residue. These non-natural residues aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems. These include, but are not limited to,peptidomimetics (e.g., chemically modified peptides, peptoids (sidechains are appended to the nitrogen atom of the peptide backbone, ratherthan to the α-carbons), β-peptides (amino group bonded to the β carbonrather than the α carbon), etc.) and other reversed or inverted forms ofamino acid moieties. Embodiments herein may, in some embodiments, belimited to natural amino acids, non-natural amino acids, and/or aminoacid analogs.

Non-conservative substitutions may involve the exchange of a member ofone class for a member from another class.

As used herein, the term “sequence identity” refers to the degree towhich two polymer sequences (e.g., peptide, polypeptide, nucleic acid,etc.) have the same sequential composition of monomer subunits. The term“sequence similarity” refers to the degree with which two polymersequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similarpolymer sequences. For example, similar amino acids are those that sharethe same biophysical characteristics and can be grouped into thefamilies (see above). The “percent sequence identity” (or “percentsequence similarity”) is calculated by: (1) comparing two optimallyaligned sequences over a window of comparison (e.g., the length of thelonger sequence, the length of the shorter sequence, a specified window,etc.), (2) determining the number of positions containing identical (orsimilar) monomers (e.g., same amino acids occurs in both sequences,similar amino acid occurs in both sequences) to yield the number ofmatched positions, (3) dividing the number of matched positions by thetotal number of positions in the comparison window (e.g., the length ofthe longer sequence, the length of the shorter sequence, a specifiedwindow), and (4) multiplying the result by 100 to yield the percentsequence identity or percent sequence similarity. For example, ifpeptides A and B are both 20 amino acids in length and have identicalamino acids at all but 1 position, then peptide A and peptide B have 95%sequence identity. If the amino acids at the non-identical positionshared the same biophysical characteristics (e.g., both were acidic),then peptide A and peptide B would have 100% sequence similarity. Asanother example, if peptide C is 20 amino acids in length and peptide Dis 15 amino acids in length, and 14 out of 15 amino acids in peptide Dare identical to those of a portion of peptide C, then peptides C and Dhave 70% sequence identity, but peptide D has 93.3% sequence identity toan optimal comparison window of peptide C. For the purpose ofcalculating “percent sequence identity” (or “percent sequencesimilarity”) herein, any gaps in aligned sequences are treated asmismatches at that position.

Any polypeptides described herein as having a particular percentsequence identity or similarity (e.g., at least 70%) with a referencesequence ID number, may also be expressed as having a maximum number ofsubstitutions (or terminal deletions) with respect to that referencesequence.

The term “effective dose” or “effective amount” refers to an amount ofan agent, e.g., a neutralizing antibody, that results in the reductionof symptoms in a patient or results in a desired biological outcome. Incertain embodiments, an effective dose or effective amount is sufficientto reduce or inhibit the infectivity of one or more strains ofinfluenza.

As used herein, the terms “administration” and “administering” refer tothe act of giving a drug, prodrug, or other agent, or therapeutic to asubject or in vivo, in vitro, or ex vivo cells, tissues, and organs.Exemplary routes of administration to the human body can be throughspace under the arachnoid membrane of the brain or spinal cord(intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical ortransdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear,rectal, vaginal, by injection (e.g., intravenously, subcutaneously,intratumorally, intraperitoneally, etc.) and the like.

The term “treatment” encompasses both therapeutic andprophylactic/preventative measures unless otherwise indicated. Those inneed of treatment include, but are not limited to, individuals alreadyhaving a particular condition (e.g., influenza infection) as well asindividuals who are at risk of acquiring a particular condition ordisorder (e.g., those needing prophylactic/preventative measures, thoseat risk of influenza exposure, those at risk of having particularly badoutcomes from influenza infection, etc.). The term “treating” refers toadministering an agent to a subject for therapeutic and/orprophylactic/preventative purposes.

A “therapeutic agent” refers to an agent that may be administered invivo to bring about a therapeutic and/or prophylactic/preventativeeffect.

A “therapeutic antibody” refers to an antibody that may be administeredin vivo to bring about a therapeutic and/or prophylactic/preventativeeffect.

As used herein, the terms “co-administration” and “co-administering”refer to the administration of at least two agent(s) or therapies to asubject. In some embodiments, the co-administration of two or moreagents or therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents or therapies lowers the requisite dosageof a potentially harmful (e.g., toxic) agent(s), and/or whenco-administration of two or more agents results in sensitization of asubject to beneficial effects of one of the agents via co-administrationof the other agent.

As used herein, the term pharmaceutical composition” refers to thecombination of an active agent (e.g., binding agent) with a carrier,inert or active, making the composition especially suitable fordiagnostic or therapeutic use in vitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see, e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference in its entirety.

DETAILED DESCRIPTION

Provided herein are anti-neuraminidase agents useful for neutralizationof influenza virus, and methods of use and manufacture thereof. Inparticular, compositions comprising anti-neuraminidase agents (e.g.,antibodies) that are cross-reactive with multiple influenza strains areprovided, as well as methods of treatment and prevention of influenzainfection therewith.

Antibodies to the hemagglutinin (HA) and neuraminidase (NA)glycoproteins are the major mediators of protection against influenzavirus infection. Experiments conducted during development of embodimentsherein demonstrate that available influenza vaccines poorly display keyNA epitopes and rarely induce NA-reactive B cells. Conversely, influenzavirus infection induces NA-reactive B cells at a frequency thatapproaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells.NA-reactive antibodies display broad binding activity spanning theentire history of influenza A virus circulation in humans, including theoriginal pandemic strains of both H1N1 and H3N2 subtypes. The antibodiesrobustly inhibit the enzymatic activity of NA, includingoseltamivir-resistant variants, and provide robust prophylacticprotection in vivo, including against avian H5N1 viruses. When usedtherapeutically, NA-reactive antibodies protected mice from lethalinfluenza virus challenge even 48-hours post-infection. These findingsindicate that influenza vaccines optimized to improve targeting of NAprovide durable and broad protection against divergent influenzastrains.

NA is an important target for antivirals or therapeutics, due to itscritical role in the influenza virus replication cycle (Wohlbold andKrammer, 2014; herein incorporated by reference in its entirety).Inhibition of NA activity is the basis of commonly used influenzatherapeutics including oseltamivir (TAMIFLU), zanamivir (RELENZA),laninamivir (INAVIR), and peramivir (RAPIVAB). Oseltamivir reduces themedian duration of influenza illness by 1.3 days and markedly reducessymptoms compared to placebo if given within 48 hours of symptom onset.In a prophylactic study, oseltamivir decreased rates of influenzainfection five-fold from 5% (25/519) for the placebo group to 1% (6/520)for the oseltamivir-treated group (Genentech, 2016; herein incorporatedby reference in its entirety). Thus, inhibition of NA activity hasbecome a standard of care for the treatment of influenza virusinfections. The limitations of neuraminidase inhibitors such asoseltamivir are that resistant strains of influenza virus have readilyemerged (Dharan et al., 2009; herein incorporated by reference in itsentirety) and the window for efficacy is limited to the first 48 hoursof symptom onset. There are several mechanisms of NA-reactive antibodyinhibition of influenza virus infection (Krammer and Palese, 2015;herein incorporated by reference in its entirety). NA-reactiveantibodies bind to influenza virus infected cells and prevent virusbudding and viral egress. These antibodies similarly inhibit viralescape from the natural defense proteins that trap the virus viaHA-sialic acid interactions on mucosal surfaces. Moreover, NA-reactiveantibody bound to NA at the surface of infected cells aids in theclearance of the virus through antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Wan etal., 2013; Wohlbold et al., 2017; herein incorporated by reference intheir entireties). The polyclonal antibody response to NA is broadlyreactive and confers protection against heterologous viruses in mice(Schulman et al., 1968; herein incorporated by reference in itsentirety). This cross-reactivity is evident even when there issubstantial change within strain specific NA epitopes, resulting in aphenomenon of one-way drift (Sandbulte et al., 2011; herein incorporatedby reference in its entirety). NA-reactive monoclonal antibodies (mAbs)isolated from mice and rabbits protected against both homologous andheterologous influenza infection in vivo (Doyle et al., 2013; Wan etal., 2013; Wan et al., 2015; Wilson et al., 2016; Wohlbold et al., 2017;herein incorporated by reference in their entireties). Several conservedamino acids were identified in these studies as the basis for the broadreactivity of NA-reactive mAbs against influenza A or B viruses (Wan etal., 2013; Wohlbold et al., 2017; herein incorporated by reference intheir entireties). Studies in humans have also shown that pre-existingNA-reactive antibodies reduce the number of cases of infection anddecrease disease severity from a naturally circulating virus (Monto andKendal, 1973; Murphy et al., 1972; herein incorporated by reference inits entirety). However, little is known about human antibody responsesto NA, and most influenza vaccine development efforts both past andpresent are focused on targeting HA.

Experiments conducted during development of embodiments hereindemonstrate that, unlike vaccination, natural influenza virus infectionreadily induces a high proportion of NA-reactive B cells. Thus, frominfected patients, protective antibodies that bind NA epitopes wereisolated and characterized, informing on the design of an NA-basedcomponent for influenza vaccination. The NA-reactive antibodies areinducible in human or mouse by infection or immunization with wholevirions, but bind epitopes not efficiently detected in the FLUARIX orFLUZONE influenza vaccines. These NA-reactive mAbs bind a broad spectrumof influenza virus strains, often spanning the entire circulationhistory in humans for that NA group. Moreover, these antibodies haverobust NA inhibition (NI) activity and provide prophylactic as well astherapeutic protection in vivo. Experiments conducted during developmentof embodiments herein provide next-generation influenza vaccines shouldthat are optimized to improve the NA humoral immune response to inducebroadly cross-reactive and protective NA-reactive antibody responses.

The results presented herein demonstrate that NA induces a potent,broadly cross-reactive, and protective humoral immune response (e.g.,with the right immunogen). The NA-reactive mAbs were more broadlyreactive, the potency of protection and neutralization rivaled that ofHA-reactive mAbs, and for H3N2 infections there were more NA-reactivethan HA-reactive B cells activated. This response is consistent with arecent report that by molar composition, NA is the most immunogenicinfluenza protein (Angeletti and Yewdell, 2017; herein incorporated byreference in its entirety). The relative conservation of NA epitopes(Sandbulte et al., 2011; herein incorporated by reference in itsentirety) also drives a back-boost effect against NAs of historicalisolates (Rajendran et al., 2017; herein incorporated by reference inits entirety). In contrast, after vaccination, experiments conductedduring development of embodiments herein demonstrate that there is onlya 1:87 ratio of NA to HA plasmablasts activated (FIG. 1E). TheNA-reactive mAbs induced by infection reported here have substantiallyreduced binding to the inactivated vaccines tested, indicating that thevaccines do not efficiently present important conserved and protectiveNA epitopes. This observation is explained by several factors. Firstly,the inactivated influenza vaccines are optimized only for the HAantigen, as the FDA requires that licensed influenza virus vaccinescontain at least 15 μg of each HA subtype (Air, 2012; hereinincorporated by reference in its entirety). Secondly, antigeniccompetition between HA and NA may affect the NA humoral immune response(Johansson et al., 1987; herein incorporated by reference in itsentirety). However, this mechanism did not appear to preclude theresponse to NA during infection or to whole virions in mice as reportedabove. Thirdly, although influenza vaccine compositions contain varyingamounts of NA (Wohlbold et al., 2015; herein incorporated by referencein its entirety), it is unclear if the NA antigen retains its naturaltetramer structure, which is important to maintain immunogenicity(Johansson and Cox, 2011; herein incorporated by reference in itsentirety). Conversely, during an influenza virus infection, NAreplicates along with the virus so that B cells can respond to intact NAon whole virions and infected cells.

The rate of NA antigenic drift is slower than that of HA, which explainsthe high frequency of broadly cross-reactive antibodies (Sandbulte etal., 2011; herein incorporated by reference in its entirety). TheNA-reactive mAbs isolated herein typically cross-bind to heterologous NAproteins from most human influenza A virus strains and a subset alsobound to avian H5N1, H7N9 and had reactivity to H7N3, H4N4, and H3N8strains. This breadth was evident for the antibodies that were used tomap the epitopes. On N1, two of the primary amino acids targeted (N309and N273) are 99.7% conserved (present in 6835 of 6855 strains) in H1N1virus from 1918 to 2017 H1N1 strain in the United States (www.fludb.org;herein incorporated by reference in its entirety). Also, N1-reactivemAbs that selected changes at two conserved epitopes (G249 and N273)shared between the human and avian strains were able to mediateprophylactic protection against H5N1 challenge in vivo in mice. Five ofthe N2-reactive mAbs bind to the conserved enzymatic active site on thehead of the NA. The broad reactivity and conservation of the targetedepitopes suggest that NA may be an essential component of universalinfluenza virus vaccine compositions.

Both NA-inhibiting and non-inhibiting mAbs to either N1 or N2 protectedfrom influenza virus challenge in vivo. Inhibition of viral egress frominfected cells or inhibition of release from mucins are the appreciatedmechanisms of action of NA-inhibiting antibodies (Krammer and Palese,2015; herein incorporated by reference in its entirety). For non-NImAbs, there are several mechanisms that account for protection. Fc-FcRinteractions have been shown to be required for full protection by someNA-reactive mAbs (DiLillo et al., 2016; Henry Dunand et al., 2016;Wohlbold et al., 2017; herein incorporated by reference in theirentireties). Although not all of the protective NA-reactive mAbs wereneutralizing in vitro, most had some degree of NA-inhibiting activity.Thus, the NA-reactive mAbs may also alter the functional balance ofopposing actions between HA and NA to disrupt efficient viralreplication (Benton et al., 2015; Wagner et al., 2002; hereinincorporated by reference in their entireties).

In some cases, infection with influenza virus induces broader and longerlasting protection than vaccination (Margine et al., 2013a; Nachbagaueret al., 2017; Wrammert et al., 2011; herein incorporated by reference intheir entireties). NA inhibiting antibody titers are recognized as acorrelate of protection (Clements et al., 1986; herein incorporated byreference in its entirety). Adult influenza virus challenge studiesshowed that antibodies inhibiting NA but not HA are associated withreduced severity and duration of illness (Memoli et al., 2016 hereinincorporated by reference in its entirety). This observation explainswhy HA and NA inhibiting antibodies are independent correlates ofvaccine effectiveness (Monto et al., 2015; herein incorporated byreference in its entirety). Experiments conducted during development ofembodiments herein demonstrate that part of such protection is mediatedby polyclonal NA-reactive antibodies that are not efficiently induced byvaccination.

There are obstacles to exploiting the broadly cross-reactive andprotective response to NA for improving influenza virus vaccines. Theimmunogenicity of NA is strain-dependent (Sultana et al., 2014; hereinincorporated by reference in its entirety) and the stability of NAs ofeach of the vaccine strains differ when subjected to variousdestabilizing agents. Using recombinant NA to induce an NA-based immuneresponse is one solution (Krammer and Palese, 2015; herein incorporatedby reference in its entirety), but the NA immunogens need to be intetrameric form for optimal immunogenicity. It is challenging to keepthe native structure of NA within vaccine formulations (Brett andJohansson, 2006; Eichelberger and Wan, 2015; herein incorporated byreference in their entireties). Another solution is the use oflive-attenuated vaccines that express NA on their surface and thesurface of infected cells. The findings described herein demonstratethat optimized NA content and structural integrity in influenza vaccinesinduces a broadly cross-reactive and protective anti-NA response.

NA-reactive antibodies are readily or even dominantly induced,protecting levels comparable to HA-reactive antibodies, but withincreased breadth. The data presented herein indicates that inclusion ofan improved NA component to influenza vaccine compositions reduced theseverity of infections. In some embodiments, the degree of protectionconferred protects across most (e.g., all) influenza infectionsoccurring at all, and in certain embodiments provides broad-rangingprotection against pandemic strains that express, for example, N1 or N2NAs.

Some embodiments described herein relate to antibodies, and antigenbinding fragments thereof, that specifically bind to epitopes on the NAprotein (e.g., N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11) of one ormore strains of influenza. Embodiments also relate to nucleic acids thatencode, immortalized B cells and cultured single plasma cells thatproduce, and to epitopes that bind, to such antibodies and antibodyfragments. In some embodiments, provided herein are vaccines comprisingthe antibodies and antigen binding fragments described herein. Inaddition, described herein is the use of the antibodies, antibodyfragments, and epitopes in screening methods as well as in thediagnosis, treatment and prevention of influenza virus infection.

In an exemplary embodiment, an antibody or an antibody fragment thereofis provided that binds an epitope on two or more (e.g., 3, 4, 5, 6, 7,8, 9, 10, 11, or ranges therebetween) NA types (e.g., N1, N2, N3, N4,N5, N6, N7, N8, N9, N10, N11) and thereby treats or prevents infectionby two or more types of influenza virus (e.g. H1N1, H3N2, H5N1, H7N1,H7N7, H9N2, etc.). Treatment/prevention of infection by other exemplarycombinations of subtypes of influenza A virus is also provided.

In some embodiments, an antibody or antibody fragment comprises a heavychain variable region having an amino acid sequence that is about 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical (or any rangestherein) to the sequence recited in any one of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 209, 217, or 225. In someembodiments, an antibody or antibody fragment comprises a heavy chainvariable regionhaving >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100% sequence similarity (or any ranges therein) to one of SEQ IDNOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 209, 217,or 225. In another embodiment, an antibody or antibody fragment of theinvention comprises a light chain variable region having an amino acidsequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical (or any ranges therein) to the sequence recited in SEQ IDNOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 213,221, or 229. In some embodiments, an antibody or antibody fragmentcomprises a light chain variable regionhaving >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100% sequence similarity (or any ranges therein) to one of SEQ IDNOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 213,221, or 229.

In some embodiments, an antibody or antibody fragment exhibits all or aportion of the epitope binding affinity of one of 228-14-035-2D04,229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06,235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06,294-16-009-A-1D05, 294-16-009-G-1F01, 296-16-003-G-2F04,300-16-005-G-2A04, 229-1D02, 229-1F06, and/or 229-2D03. In someembodiments, an antibody or antibody fragment binds the same epitope asone of 228-14-035-2D04, 229-14-036-1D05, 229-14-036-1G03,229-14-036-2B04, 229-14-036-2C06, 235-15-042-1E06, 1000-2E06,294-16-009-A-1C02, 294-16-009-A-1C06, 294-16-009-A-1D05,294-16-009-G-1F01, 296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02,229-1F06, and/or 229-2D03. In some embodiments, an antibody or antibodyfragment exhibits the influenza neutralizing activity of one of228-14-035-2D04, 229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04,229-14-036-2C06, 235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02,294-16-009-A-1C06, 294-16-009-A-1D05, 294-16-009-G-1F01,296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02, 229-1F06, and/or229-2D03. In some embodiments, an antibody or antibody fragmentneutralizes the same influenza strains as one of 228-14-035-2D04,229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06,235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06,294-16-009-A-1D05, 294-16-009-G-1F01, 296-16-003-G-2F04,300-16-005-G-2A04, 229-1D02, 229-1F06, and/or 229-2D03. In someembodiments, an antibody is not a natural antibody. In some embodiments,an antibody is not a natural human antibody.

The CDRs of the antibody heavy chains are referred to as CDRH1 (orHCDR1), CDRH2 (or HCDR2) and CDRH3 (or HCDR3), respectively. Similarly,the CDRs of the antibody light chains are referred to either as CDRK1(or KCDR1), CDRK2 (or KCDR1) and CDRK3 (or KCDR1), or CDRL1 (or LCDR1),CDRL2 (or LCDR1) and CDRL3 (or LCDR1), respectively. In someembodiments, antibodies or antibody fragments are provided with heavychain CDR1 corresponding to one of SEQ ID NOs: 4, 20, 36, 52, 68, 84,100, 116, 132, 148, 164, 180, or 196. In some embodiments, antibodies orantibody fragments are provided with heavy chain CDR2 corresponding toone of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182,198. In some embodiments, antibodies or antibody fragments are providedwith heavy chain CDR3 corresponding to one of SEQ ID NOs: 8, 24, 40, 56,72, 88, 104, 120, 136, 152, 168, 184, 200. In some embodiments,antibodies or antibody fragments are provided with light chain CDRscorresponding to one or SEQ ID NOs: 22-24, 25-27, 28-30, or 40-42. Insome embodiments, antibodies or antibody fragments are provided withlight chain CDR1 corresponding to one of SEQ ID NOs: 12, 28, 44, 60, 76,92, 108, 124, 140, 156, 172, 188, 204. In some embodiments, antibodiesor antibody fragments are provided with light chain CDR2 correspondingto one of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174,190, 206. In some embodiments, antibodies or antibody fragments areprovided with light chain CDR3 corresponding to one of SEQ ID NOs: 16,32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208. In someembodiments, CDRs are provided having at least 70% sequence identity(e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, and any ranges withsuch endpoints (e.g., 70-100%, 80-100%, 85-99%, 90-99%, etc.)) with oneof SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196,6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 8, 24, 40, 56,72, 88, 104, 120, 136, 152, 168, 184, 200, 12, 28, 44, 60, 76, 92, 108,124, 140, 156, 172, 188, 204, 14, 30, 46, 62, 78, 94, 110, 126, 142,158, 174, 190, 206, 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176,192, and/or 208. In some embodiments, CDRs are provided having at least50% sequence similarity (e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, 100%, and any ranges with such endpoints (e.g., 50-100%,80-100%, 85-99%, 90-99%, etc.)) with one of SEQ ID NOs: 4, 20, 36, 52,68, 84, 100, 116, 132, 148, 164, 180, 196, 6, 22, 38, 54, 70, 86, 102,118, 134, 150, 166, 182, 198, 8, 24, 40, 56, 72, 88, 104, 120, 136, 152,168, 184, 200, 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188,204, 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 16, 32,48, 64, 80, 96, 112, 128, 144, 160, 176, 192, and/or 208. In someembodiments, CDRs (or a combination thereof) are provided that recognizethe same HA epitopes as 228-14-035-2D04, 229-14-036-1D05,229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06, 235-15-042-1E06,1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06, 294-16-009-A-1D05,294-16-009-G-1F01, 296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02,229-1F06, and/or 229-2D03.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 228-14-035-2D04 (SEQ ID NOs: 4, 6, 8, 12, 14, and 16), andneutralizes influenza virus infection. In some embodiments, an antibodyor antigen binding fragment comprises CDRs with at least 70% sequenceidentity (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or100%, and ranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 228-14-035-2D04(SEQ ID NOs: 4, 6, 8, 12, 14, and 16), binds the epitope(s) of antibody228-14-035-2D04, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises all of the CDRs of antibody 229-14-036-1D05 (SEQ ID NOs: 20,22, 24, 28, 30, and 32), and neutralizes influenza virus infection. Insome embodiments, an antibody or antigen binding fragment comprises CDRswith at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-14-036-1D05(SEQ ID NOs: 20, 22, 24, 28, 30, and 32), binds the epitope(s) ofantibody 229-14-036-1D05, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises all of the CDRs of antibody 229-14-036-1G03 (SEQ ID NOs: 36,38, 40, 44, 46, and 48), and neutralizes influenza virus infection. Insome embodiments, an antibody or antigen binding fragment comprises CDRswith at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-14-036-1G03(SEQ ID NOs: 36, 38, 40, 44, 46, and 48), binds the epitope(s) ofantibody 229-14-036-1G03, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises all of the CDRs of antibody 229-14-036-2B04 (SEQ ID NOs: 52,54, 56, 60, 62, and 64), and neutralizes influenza virus infection. Insome embodiments, an antibody or antigen binding fragment comprises CDRswith at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-14-036-2B04(SEQ ID NOs: 52, 54, 56, 60, 62, and 64), binds the epitope(s) ofantibody 229-14-036-2B04, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises all of the CDRs of antibody 229-14-036-2C06 (SEQ ID NOs: 68,70, 72, 76, 78, and 80), and neutralizes influenza virus infection. Insome embodiments, an antibody or antigen binding fragment comprises CDRswith at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-14-036-2C06(SEQ ID NOs: 68, 70, 72, 76, 78, and 80), binds the epitope(s) ofantibody 229-14-036-2C06, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 235-15-042-1E06 (SEQ ID NOs: 84, 86, 88, 92, 94, and 96), andneutralizes influenza virus infection. In some embodiments, an antibodyor antigen binding fragment comprises CDRs with at least 70% sequenceidentity (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or100%, and ranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 235-15-042-1E06(SEQ ID NOs: 84, 86, 88, 92, 94, and 96), binds the epitope(s) ofantibody 235-15-042-1E06, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 1000-2E06 (SEQ ID NOs: 100, 102, 104, 108, 110, and 112), andneutralizes influenza virus infection. In some embodiments, an antibodyor antigen binding fragment comprises CDRs with at least 70% sequenceidentity (e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or100%, and ranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 1000-2E06 (SEQ IDNOs: 84, 86, 88, 92, 94, and 96), binds the epitope(s) of antibody1000-2E06, and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 294-16-009-A-1C02 (SEQ ID NOs: 116, 118, 120, 124, 126, and128), and neutralizes influenza virus infection. In some embodiments, anantibody or antigen binding fragment comprises CDRs with at least 70%sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 294-16-009-A-1C02(SEQ ID NOs: 116, 118, 120, 124, 126, and 128), binds the epitope(s) ofantibody 294-16-009-A-1C02, and/or neutralizes influenza virusinfection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 294-16-009-A-1C06 (SEQ ID NOs: 132, 134, 136, 140, 142, and144), and neutralizes influenza virus infection. In some embodiments, anantibody or antigen binding fragment comprises CDRs with at least 70%sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 294-16-009-A-1C06(SEQ ID NOs: 132, 134, 136, 140, 142, and 144), binds the epitope(s) ofantibody 294-16-009-A-1C06, and/or neutralizes influenza virusinfection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 294-16-009-A-1D05 (SEQ ID NOs: 148, 150, 152, 156, 158, and160), and neutralizes influenza virus infection. In some embodiments, anantibody or antigen binding fragment comprises CDRs with at least 70%sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 294-16-009-A-1D05(SEQ ID NOs: 148, 150, 152, 156, 158, and 160), binds the epitope(s) ofantibody 294-16-009-A-1D05, and/or neutralizes influenza virusinfection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 294-16-009-G-1F01 (SEQ ID NOs: 164, 166, 168, 172, 174, and176), and neutralizes influenza virus infection. In some embodiments, anantibody or antigen binding fragment comprises CDRs with at least 70%sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 294-16-009-G-1F01(SEQ ID NOs: 164, 166, 168, 172, 174, and 176), binds the epitope(s) ofantibody 294-16-009-G-1F01, and/or neutralizes influenza virusinfection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 296-16-003-G-2F04 (SEQ ID NOs: 180, 182, 184, 188, 190, and192), and neutralizes influenza virus infection. In some embodiments, anantibody or antigen binding fragment comprises CDRs with at least 70%sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 296-16-003-G-2F04(SEQ ID NOs: 180, 182, 184, 188, 190, and 192), binds the epitope(s) ofantibody 296-16-003-G-2F04, and/or neutralizes influenza virusinfection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 300-16-005-G-2A04 (SEQ ID NOs: 196, 198, 200, 204, 206, and208), and neutralizes influenza virus infection. In some embodiments, anantibody or antigen binding fragment comprises CDRs with at least 70%sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 300-16-005-G-2A04(SEQ ID NOs: 196, 198, 200, 204, 206, and 208), binds the epitope(s) ofantibody 300-16-005-G-2A04, and/or neutralizes influenza virusinfection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 229-1D02 (SEQ ID NOs: 210-212 and 214-216), and neutralizesinfluenza virus infection. In some embodiments, an antibody or antigenbinding fragment comprises CDRs with at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-1D02 (SEQ IDNOs: 210-212 and 14-216), binds the epitope(s) of antibody 229-1D02,and/or neutralizes influenza virus infection. 229-1D02 exhibits lowaffinity binding toward the recent H1N1 strains, A/California/2009(Kd=2.316×10{circumflex over ( )}-8) and A/Brisbane/2007(Kd=1.893×10{circumflex over ( )}-8). Such heterosubtypic binding of NAantibodies is rare. Binding curves for 229 1D02 against several H1N1 andH3N2 strains are depicted in FIG. 11.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 229-1F06 (SEQ ID NOs: 218-220 and 222-224), and neutralizesinfluenza virus infection. In some embodiments, an antibody or antigenbinding fragment comprises CDRs with at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-1F06 (SEQ IDNOs: 218-220 and 222-224), binds the epitope(s) of antibody 229-1F06,and/or neutralizes influenza virus infection.

In certain embodiments, an antibody or antigen binding fragmentcomprises the light chain CDRs, heavy chain CDRs, or all of the CDRs ofantibody 229-2D03 (SEQ ID NOs: 226-218 and 230-232), and neutralizesinfluenza virus infection. In some embodiments, an antibody or antigenbinding fragment comprises CDRs with at least 70% sequence identity(e.g., >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99% or 100%, andranges therein) and/or at least 50% sequence similarity(e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >97%, >98%, >99%or 100%, and ranges therein) with the CDRs of antibody 229-2D03 (SEQ IDNOs: 226-218 and 230-232), binds the epitope(s) of antibody 229-2D03,and/or neutralizes influenza virus infection.

In some embodiments, an antibody or antigen binding fragment comprisesless than 100% sequence identity with the light chain, heavy chain, orall of any of the antibody sequences of 228-14-035-2D04,229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06,235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06,294-16-009-A-1D05, 294-16-009-G-1F01, 296-16-003-G-2F04,300-16-005-G-2A04, 229-1D02, 229-1F06, and/or 229-2D03. In someembodiments, an antibody or antigen binding fragment comprises less than100% sequence identity with SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58,66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146, 154, 162, 170, 178,186, 194, 209, 213, 217, 221, 225, and/or 229.

The invention further comprises an antibody, or fragment thereof, thatbinds to the same epitope as an antibody described herein (e.g.,228-14-035-2D04, 229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04,229-14-036-2C06, 235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02,294-16-009-A-1C06, 294-16-009-A-1D05, 294-16-009-G-1F01,296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02, 229-1F06, and/or229-2D03), or an antibody that competes with an antibody or antigenbinding fragment described herein.

Antibodies within the scope described herein may also include hybridantibody molecules that comprise one or more CDRs from an antibodydescribed herein (e.g., 228-14-035-2D04, 229-14-036-1D05,229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06, 235-15-042-1E06,1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06, 294-16-009-A-1D05,294-16-009-G-1F01, 296-16-003-G-2F04, 300-16-005-G-2A04, 229-1D02,229-1F06, and/or 229-2D03) and one or more CDRs from another antibody tothe same epitope. In one embodiment, such hybrid antibodies comprisethree CDRs from an antibody described herein and three CDRs from anotherantibody to the same epitope. Exemplary hybrid antibodies comprise: (i)the three light chain CDRs from an antibody described herein and thethree heavy chain CDRs from another antibody to the same epitope, or(ii) the three heavy chain CDRs from an antibody described herein andthe three light chain CDRs from another antibody to the same epitope.

Variant antibodies are also included within the scope herein. Thus,variants of the sequences recited in the application are also includedwithin the scope herein. Such variants include natural variantsgenerated by somatic mutation in vivo during the immune response or invitro upon culture of immortalized B cell clones. Alternatively,variants may arise due to the degeneracy of the genetic code, or may beproduced due to errors in transcription or translation.

Further variants of the antibody sequences having improved affinityand/or potency may be obtained using methods known in the art and areincluded within the scope herein. For example, amino acid substitutionsmay be used to obtain antibodies with further improved affinity.Alternatively, codon optimization of the nucleotide sequence may be usedto improve the efficiency of translation in expression systems for theproduction of the antibody. Further, polynucleotides comprising asequence optimized for antibody specificity or neutralizing activity bythe application of a directed evolution method to any of the nucleicacid sequences here are also within the scope included herein.

In some embodiments, variant antibody sequences may share 70% or more(e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more, or rangestherein) amino acid sequence identity with the sequences recited herein.In some embodiments, variant antibody sequences may share 50% or more(e.g., 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more, orranges therein) amino acid sequence similarity with the sequencesrecited herein.

In one embodiment, nucleic acid sequences described herein includenucleic acid sequences having at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identity to the nucleic acid encoding a heavy or light chain of anantibody described herein (e.g., SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97,113, 129, 145, 161, 177, 193, 9, 25, 41, 57, 73, 89, 105, 121, 137, 153,169, 185, and/or 201). In another embodiment, a nucleic acid sequencehas the sequence of a nucleic acid encoding a heavy or light chain CDRof an antibody of the invention (e.g., SEQ ID NOs: 3, 19, 35, 51, 67,83, 99, 115, 131, 147, 163, 179, 195, 5, 21, 37, 53, 69, 85, 101, 117,133, 149, 165, 181, 197, 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167,183, 199, 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 13,29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 15, 31, 47, 63,79, 95, 111, 127, 143, 159, 175, 191, and/or 207).

In some embodiments, provided herein are modified antibodies and/ormodified antibody fragments (e.g., antibodies and antibody fragmentscomprising non-natural amino acids, substituents, bonds, moieties,connections, etc.). For example, modifications may comprise theintroduction of disulfide bonds, glycosylation, lipidation, acetylation,phosphorylation, or any other manipulation or modification, such asconjugation with a labeling or therapeutic agent. Modifications may alsoinclude the substitution of natural amino acids for amino acid analogs(including, for example, unnatural amino acids, etc.), as well as othermodifications known in the art.

In some embodiments, an antibody finding use in embodiments herein is anon-natural immunogenic agent, such as: an antibody fragment, anon-natural antibody comprising the CDRs herein, a modified antibody, amonoclonal antibody, a humanized antibody, a chimeric antibody, andnon-natural combinations thereof.

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid sequencedescribed herein. Cells transformed with such vectors are also included.Examples of such cells include but are not limited to, eukaryotic cells,e.g. yeast cells, animal cells or plant cells. In one embodiment thecells are mammalian, e.g. human, CHO, HEK293T, PER.C6, NS0, myeloma orhybridoma cells.

Embodiments within the scope of this disclosure include methods ofpreventing or treating influenza infections comprising administering atherapeutically-effective or prophylactically effective amount of amonoclonal antibody having specificity for an NA epitope. In someembodiments, an antibody recognizes (e.g., has affinity and/orspecificity for) epitopes having at least 90%, at least 92%, at least95%, at least 97%, at least 98%, or at least 99% homology to epitope(s)recognized by (e.g., has affinity and/or specificity for) the antibodiesdescribed herein.

In some embodiments, a pharmaceutical composition comprising theantibodies disclosed herein includes an acceptable carrier and isformulated into a suitable dosage form according to administrationmodes. Pharmaceutical preparations suitable for administration modes areknown, and generally include surfactants that facilitate transportacross the membrane. Such surfactants may be derived from steroids, ormay be cationic lipids such asN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),or various compounds such as cholesterol hemisuccinate and phosphatidylglycerol.

For oral administration, the pharmaceutical composition may be presentedas discrete units, for example, capsules or tablets; powders orgranules; solutions, syrups or suspensions (edible foam or whipformulations in aqueous or non-aqueous liquids); or emulsions.

For parenteral administration, the pharmaceutical composition mayinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients availablefor use in injectable solutions include, for example, water, alcohol,polyols, glycerin, and vegetable oils. Such a composition may bepresented in unit-dose (single dose) or multiple dose (several doses)containers, for example, sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical composition may include antiseptics, solubilizers,stabilizers, wetting agents, emulsifiers, sweeteners, colorants,odorants, salts, buffering agents, coating agents, or anti-oxidants.

Compositions may comprise, in addition to the antibody or antibodiesdescribed herein, a therapeutically active agent (e.g., drug),additional antibodies (e.g., against influenza or another target), etc.

The present composition may be formulated into dosage forms for use inhumans or veterinary use. The composition comprising the antibodie(s)may be administered to influenza-infected or highly susceptible humansand livestock, such as cows, horses, sheep, swine, goats, camels, andantelopes, in order to prevent or treat the incidence of influenza. Whena subject is already infected, the present antibodie(s) may beadministered alone or in combination with another antiviral treatment.

The antibody composition may be administered in a pharmaceuticallyeffective amount in a single- or multiple-dose. The pharmaceuticalcomposition may be administered via any of the common routes, as long asit is able to reach the desired tissue. Thus, the present compositionmay be administered via oral or parenteral (e.g., subcutaneous,intramuscular, intravenous, or intradermal administration) routes, andmay be formulated into various dosage forms. In one embodiment, theformulation is an injectable preparation. Intravenous, subcutaneous,intradermal, intramuscular and dropping injectable preparations arepossible.

Antibodies may be coupled to a drug for delivery to a treatment site orcoupled to a detectable label to facilitate imaging of a site comprisingcells of interest, such as cells infected with influenza A virus.Methods for coupling antibodies to drugs and detectable labels are wellknown in the art, as are methods for imaging using detectable labels.Labeled antibodies may be employed in a wide variety of assays,employing a wide variety of labels. Detection of the formation of anantibody-antigen complex between an antibody of the invention and anepitope of interest (an influenza A virus epitope) can be facilitated byattaching a detectable substance to the antibody. Suitable detectionmeans include the use of labels such as radionuclides, enzymes,coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substratesor co-factors, enzyme inhibitors, prosthetic group complexes, freeradicals, particles, dyes, and the like. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material isluminol; examples of bioluminescent materials include luciferase,luciferin, and acquorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H. Such labeled reagents may be used in avariety of well-known assays, such as radioimmunoassays, enzymeimmunoassays, e.g., ELISA, fluorescent immunoassays, and the like.

An antibody may be conjugated to a therapeutic moiety. Such antibodyconjugates can be used for modifying a given biological response; thedrug moiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Techniques forconjugating such therapeutic moiety to antibodies are well known. See,for example, Arnon et al. (1985) “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy,” in Monoclonal Antibodiesand Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp.243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,” inControlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurds, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158; herein incorporated by reference intheir entireties.

Alternatively, an antibody, or antibody fragment thereof, can beconjugated to a second antibody, or antibody fragment thereof, to forman antibody heteroconjugate as described in U.S. Pat. No. 4,676,980;herein incorporated by reference in its entirety. In addition, linkersmay be used between the labels and the antibodies of the invention (e.g.U.S. Pat. No. 4,831,175; herein incorporated by reference in itsentirety).

Antibodies of the invention may also be attached to a solid support.Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life. In someembodiments the polymers may be selected from polyoxyethylated polyolsand polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH2-CH2)n O—R where R canbe hydrogen, or a protective group such as an alkyl or alkanol group.

Water-soluble polyoxyethylated polyols may also be employed. Theyinclude polyoxyethylated sorbitol, polyoxyethylated glucose,polyoxyethylated glycerol (POG), and the like. Another drug deliverysystem that can be used for increasing circulatory half-life is theliposome.

Antibodies may be provided in purified form. Typically, the antibodywill be present in a composition that is substantially free of otherpolypeptides e.g. where less than 90% (by weight), usually less than 60%and more usually less than 50% of the composition is made up of otherpolypeptides.

Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgM(e.g., an alpha, gamma or mu heavy chain). Within the IgG isotype,antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass. Antibodies may havea kappa or a lamda light chain.

EXPERIMENTAL Example 1 Materials and Methods Cell, Viruses andRecombinant Proteins

Human embryo kidney (HEK) 293T and Madin-Darby canine kidney (MDCK)cells were obtained from the American Type Culture Collection (ATCC).All influenza virus stocks used for the assays were freshly grown inspecific pathogen free (SPF) eggs, harvested, purified and titered. Areassortant H6N2 virus with the backbone from A/Puerto Rico/8/34 (PR8)containing the HA gene of A/turkey/Massachusetts/3740/76 and the NA fromA/Minnesota/11/2010 was used to generate the mutant viruses (S153T,N199K, N221K, G248E, S322F, K344E, G346D, E369T, K400R, G429E, K435E andW437R single mutation in the NA gene). A/Switzerland/9715293/2013 (H3N2)was treated with 0.02% formaldehyde for 48 h to generate the inactivevirus particles. The inactivation was verified by injecting treatedvirus into eggs followed by HA measurements. Recombinant NA proteinsderived from A/Puerto Rico/8/1934 (H1N1), A/New Caledonia/20/1999(H1N1), A/Brisbane/59/2007 (H1N1), A/California/7/2009 (H1N1), A/greyteal/Australia/2/1979 (H4N4), A/Shanghai/1/2013 (H7N9),A/equine/Pennsylvania/1/2007 (H3N8), A/turkey/Wisconsin/1/1966 (H9N2)were obtained from BEI resources and A/Canada/444/2004 (H7N3) N3 NA wasobtained from the Influenza Reagent Resource (IRR). The otherrecombinant NA and HA proteins were expressed in-house, in a baculovirusexpression system (Margine et al., 2013b; herein incorporated byreference in its entirety).

Monoclonal Antibodies

Antibodies were generated as described in Smith et al., 2009; Wrammertet al., 2008; herein incorporated by reference in their entireties.Peripheral blood was obtained from each subject 7 days after infectionor vaccination. Lymphocytes were isolated and enriched for B cells usingRosetteSep. Plasmablasts (CD3− CD19+ CD20low CD27hi CD38hi) were singlecell-sorted into 96-well plates. Immunoglobulin variable genes fromplasmablasts were amplified by reverse transcriptase polymerase chainreaction (RT-PCR) and sequenced, then cloned into human IgG1 expressionvectors and co-transfected into HEK293 cells. Secreted mAbs werepurified from the supernatant using protein A beads.

Enzyme Linked Immunosorbent Assay (ELISA)

High-protein binding microtiter plates (Costar) were coated with 8hemagglutinating units (HAU) of whole virus per well or recombinant NAsor HAs at 1 μg/ml in phosphate buffered saline (PBS) overnight at 4° C.After blocking, serially diluted antibodies 1:3 starting at 10 μg/mlwere incubated for 1 h at 37° C. Horse radish peroxidase(HRP)-conjugated goat anti-human IgG antibody diluted 1:1000 (JacksonImmuno Research) was used to detect binding of mAbs, and was developedwith Super Aquablue ELISA substrate (eBiosciences). Absorbance wasmeasured at 405 nm on a microplate spectrophotometer (BioRad). Tostandardize the assays, antibodies with known binding characteristicswere included on each plate and the plates were developed when theabsorbance of the control reached 3.0 OD units. Competition ELISAs wereperformed by inhibiting binding of each biotinylated antibody ofinterest at the half-maximal binding concentration with a 10-fold molarexcess of competitor antibody. HRP conjugated streptavidin diluted1:1000 (Southern Biotech) was used for detection. Plates were developeduntil samples in the absence of competitor antibody reached an OD of 1(Henry Dunand et al., 2015; herein incorporated by reference in itsentirety).

Cell-Based ELISA

A/California/7/2009NA and its mutants were expressed on 293T cells bytransfecting with wild type or mutant pCAGGS-CA/09NA plasmids usingLipofectamine 2000 reagent (Invitrogen). ELISA was performed with thetransfected cells as described previously (Wan et al., 2013). For allother NAs (mutant and wild type), the signals generated by mAb bindingto each NA were normalized to those generated by mouse serum (thebackground signals generated with mock-transfected cells were subtractedfrom both the mAb and mouse serum signals) and therefore expressed asrelative binding.

Microneutralization Assay (MN)

MN assay for antibody characterization was carried out (Henry Dunand etal., 2015; herein incorporated by reference in its entirety). MDCK cellswere maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal bovine serum (FBS) at 37° C. with 5% CO₂. Onthe day before the experiment, confluent MDCK cells in a 96-well formatwere washed twice with PBS and incubated in minimal essential medium(MEM) supplemented with 1 μg/ml trypsin-ethylenediamine tetraacetic acid(EDTA). Serial 2-fold dilutions (starting concentration 128 μg/ml) ofmAb were mixed with an equal volume of 100 50% tissue culture infectiousdoses (TCID₅₀) virus and incubated for 1 h at 37° C. The mixture wasremoved and cells were cultured for 20 h at 37° C. with 1×MEMsupplemented with 1 μg/ml trypsin-TPCK and appropriate mAbconcentration. Cells were washed twice with PBS, fixed with 80% ice coldacetone at −20° C. for 1 h, washed 3 times with PBS, blocked for 30 minwith 10% FBS and then treated for 30 min with 2% H₂O₂. Ananti-NP-biotinylated antibody (1:3000) in 3% BSA-PBS was incubated for 1h at room temperature. The plates were developed with Super AquablueELISA substrate at 405 nm. The signal from uninfected wells wereaveraged to represent 100% inhibition. Virus infected wells without mAbwere averaged to represent 0% inhibition. Duplication wells were used tocalculate the mean and SD of neutralization, and inhibitoryconcentration 50 (IC₅₀) was determined by a sigmoidal dose responsecurve. The inhibition ratio (%) was calculated as below:

(OD(Pos. Control)−OD(Sample))/(OD(Pos. Control)−OD(Neg. Control))×100%

The final concentration of antibody that reduced infection to 50% (IC₅₀)was determined using Prism software (GraphPad).

NA Enzyme-Linked Lectin Assay (ELLA)

ELLAs were performed as described (Westgeest et al., 2015; hereinincorporated by reference in its entirety). Flat-bottom nonsterile96-well plates (Thermo Scientific) were coated with 100 μl of fetuin(Sigma) at 25 μg/ml at 4° C. overnight. 50 μl antibodies were seriallydiluted (two-fold) in Dulbecco's phosphate-buffered saline (DPBS)containing 0.133 g/L CaCl₂) and 0.1 g/L MgCl₂ with 0.05% Tween 20 and 1%BSA (DPBSTBsA), then incubated in duplicate fetuin-coated plates with anequal volume of the selected antigen dilution in DPBSTBsA. These plateswere subsequently sealed and incubated for 18 h at 37° C. The plateswere subsequently washed six times with PBS with 0.05% Tween 20, and 100μl/well of HRP-conjugated peanut agglutinin lectin (PNA-HRPO,Sigma-Aldrich) in DPBSTBsA was added for 2 h at RT in the dark. Theplates were washed six times and were developed with Super AquablueELISA substrate (eBiosciences). Absorbance was read at 405 nm on amicroplate spectrophotometer (BioRad). Data points were analyzed usingPrism software and the 50% inhibition concentration (IC50) was definedas concentration at which 50% of the NA activity was inhibited comparedto the negative control.

NA-STAR Assay

The NA-STAR assay was performed according to the Resistance DetectionKit manufacturer's instructions (Applied Biosystems, Darmstadt, Germany)(Nguyen et al., 2010; herein incorporated by reference in its entirety).25 μl test mAbs in serial two-fold dilutions in NA-Star assay buffer (26mM 2-(N-morpholino) ethanesulfonic acid; 4 mM calcium chloride; pH 6.0)were mixed with 25 μl of NA protein or 4×IC₅₀ of virus and incubated at37° C. for 20 min. After adding 10 μl of 1000× diluted NA-Starsubstrate, the plates were incubated at room temperature for 30 min. Thereaction was stopped by adding 60 μl of NA Star accelerator. Thechemiluminescent was determined by using the DTX 880 plate reader(Beckman Coulter). Data points were analyzed using Prism software andthe 50% inhibition concentration (IC₅₀) was defined as concentration atwhich 50% of the NA activity was inhibited compared to the negativecontrol.

Competition Studies Using Bio-Layer Interferometry

A fortéBio Octet K2 instrument was used to measure the competitionbetween the N2-reactive mAbs and oseltamivir. A/Texas/50/2012 rNA (5μg/ml) in PBS was used to load anti-His probes for 300 s, then theprobes were moved to oseltamivir (25 μg/ml) and control PBS for another300 s, and following by binding of the complex to the N2-reactive mAbs(50 μg/ml) for 300 s to 500 s. The final volume for all the solutionswas 200 μl/well. All of the assays were performed with agitation set to1,000 r.p.m. in PBS buffer supplemented with 1% BSA to minimizenonspecific interactions at 30° C.

Mouse Challenge and Immunization Experiments

In prophylactic studies, five female BALB/c mice (The JacksonLaboratory) per group aged 6 to 8 weeks received a 5 mg/kg dose of mAbsintraperitoneally (i.p.). After 2 h treatment, the mice wereanesthetized using a ketamine-xylazine mixture and intranasally infectedwith 10× the 50% lethal dose (LD₅₀) of A/Netherlands/602/2009 (H1N1),A/Philippines/2/1982 (H3N2, X-79-surface glycoproteins fromA/Philippines/2/1982 and backbone from A/PR/8/34) or A/Vietnam/1203/2004(H5N1-surface glycoproteins from A/Vietnam/1203/2004 and backbone fromA/PR/8/34, polybasic cleavage site replaced with a regular cleavagesite). In a therapeutic setting, mice received a 10 mg/kg dose of eachmAbs i.p. 48 h after 10 LD50 virus intranasal inoculation (in a 30 μlinoculum). In all groups, mice were monitored daily for survival andweight loss until day 14 post-infection. Mice that lost 25% or more oftheir initial body weights were euthanized. For the immunization assays,mice were infected by 0.25 LD50 of A/Netherlands/602/2009 (H1N1) orimmunized with 2 μg of inactivated A/Switzerland/9715293/2013 (H3N2)influenza virus intranasally and boosted on day 30 using the sameimmunogens/doses. Spleen cells were collected on day 38 and analyzed forthe HA and NA humoral immune response by ELISPOT.

Purification of NA-Reactive IgG from Serum

Each serum sample analyzed was passed through a 5 ml Protein G Plusagarose (Pierce) affinity column in gravity mode. Serum flow-through wascollected and passed through the column three times. The column was thenwashed with 15 column volume (CV) of PBS prior to elution with 5 CV of100 mM glycine-HCl, pH 2.7. The eluate, containing total IgG from serum,was immediately neutralized with 5 ml of 1 M Tris-HCl, pH 8.0. Theflow-through was subjected to the same purification process one moretime to capture all IgG from serum, and the two eluates were combined.To isolate the NA-reactive IgG, recombinant N2 neuraminidase (rNA) fromA/Hong Kong/4801/2014 was first biotinylated using the EZ-linkSulfo-NHS-Biotin (Thermo Scientific) according to the methods providedby the manufacturers. Biotinylated rNA was then bound to NeutrAvidinagarose resins (Pierce) packed into a 0.5 ml chromatography column(Clontech). The resins were equilibrated with 10 CV of PBS. Total IgGwas applied to a column packed with Neutravidin agarose resins only, andflow-through was collected in order to remove any resin-binding IgGs.The collected samples were then subjected to the affinity column withrNA in gravity mode, and flow-through was collected and reapplied to thecolumn three times. The column was washed with 10 CV of PBS and elutedwith 5 CV of 100 mM glycine-HCl, pH 2.7 and immediately neutralized with1 M Tris-HCl, pH 8.0. The flow-through from each pull-down was subjectedto the same purification process until all of NA-reactive IgGs wereisolated. All eluate samples from each donor were combined, thenbuffer-exchanged into PBS and concentrated using a 30 kDa Vivaspin 15centrifuge tube (Sartorius).

Statistical Analysis

Statistical analysis was performed using Prism software (Graphpad).Specific tests for statistical significance are detailed in the figurelegends. P values equal to or less than 0.05 were consideredsignificant.

Example 2 Results

NA is Frequently Targeted by Plasmablasts Activated During NaturalInfluenza Virus Infection but not after Vaccination

While characterizing the specificity of plasmablasts induced byinfluenza virus infection, a high proportion of NA-reactive cells wasobserved. The specificity of plasmablasts was evaluated by ELISPOT ormAb characterization from a total of sixteen confirmedinfluenza-infected patients.

These patients included eleven patients infected with the H1N1 pandemicstrain (five from 2009 and six from 2016), plus five patients wereinfected with H3N2 virus strains, including three in 2014 and two in2017 (clinical data is provided in Table 1). First, large numbers ofactivated plasmablasts were analyzed in six influenza virus infectedpatients (four infected with H1N1 in 2016 and two infected with H3N2 in2017). Scoring of thousands of activated plasmablasts by ELISPOT assaydetected an average of 24% that were reactive to NA and 38% to HA (FIG.1A). Plasmablasts from H3N2 infected patients predominantly targeted NA.To more rigorously assess the frequency of NA-reactive B cells activatedduring infection, mAbs obtained from patients were characterized. Theisolated variable region genes from single plasmablasts activated byinfection were used to express mAb proteins from 12 of the patients(See, e.g., Smith et al., 2009; Wardemann et al., 2003; Wrammert et al.,2008; herein incorporated by reference in their entireties). TheNA-reactive mAbs were more often encoded by VH3 family genes, but usedvariable genes that were otherwise similar to HA antibodies (FIG. 8).Consistent with the ELISPOT assays, 22.6% (29/128), and on average 24%by year and strain, of plasmablast mAbs activated by influenza virusinfection were reactive to recombinant NA (rNA) (FIGS. 1B, 1C, and 1D).As with the ELISPOT analysis, H3N2 virus infections consistently induceda higher proportion of NA-reactive B cells compared to HA-reactive Bcells for all five patients assessed (FIGS. 1A and 1D, blue dots). Bycomparison, activation of NA-reactive B cells was quite rare aftervaccination, accounting for only 1.2% (3/258) of induced plasmablastsrelative to 87% that targeted HA (FIG. 1E). This observation wasconsistent for several influenza virus vaccine compositions, including1.5% (2 of 133) of NA-reactive cells after immunization with a subunitvaccine (from 2006-2008 and in 2010), 1.1% (1 of 89) after the 2009 H1N1monovalent vaccine, and none (0 of 36) induced by split vaccines(2008-2010) (FIG. 1E). The analysis demonstrates that a quarter ofplasmablasts induced by natural influenza virus infection target NA—apercentage that nearly equals that of HA-specific plasmablasts—comparedto only 1-2% from influenza vaccination.

TABLE 1 Summary of clinical data for patients with acute influenza virusinfections YEAR ID Age Gen Influenza A Strain Vaccine HistoryComorbidities 2009 EM 30 F Pan H1N1 N/A NONE 2009 1000 37 M Pan H1N1 N/AHypertension, interstitial lung disease of unknown

ology 2009  70 38 F Pan H1N1 N/A NONE 2009 1009 21 M Pan H1N1 N/ACongenital heart disease,

 for tetralogy of Fallot 2009 1011 2

M Pan H1N1 N/A NONE 2016 294-16- 2

M Pan H1N1 2015 NONE 009 2016 294-16- 26 M Pan H1N1 No NONE 003 History201

00

-14- 24 F Pan H1N1 N/A NONE 01

1 201

R018-14- 43 F Pan H1N1 N/A NONE 0101 2016 300-16- 30 M Pan H1N1 No NONE00

History 2016 301-16- 46 F Pan H1N1 2014 Hypertention, asthma 007 2014228-14- 34 M S H1N2 2011- ASTHMA 0

5 2013 2014 229-14- 46 F S H1N2 2011- COPD ASTHMA 03

2013 2014 235-15- 4

M S H3N2 2009- OA, ASTHMA, CHF 042 2014 2017

19-17- 38 M S H3N2 2013- NONE 008 2014 2017 32

-17- 31 M S H3N2 No NONE 012 History Anti- Initial Hospital Sample viralYEAR Symptoms* course collection treatment 2009 Dyspnea Acuterespiratory distress

, bacterial D

1

,

 prolonged

 support,

, discharged

 2 months 2009 Shortness of breath,

, acute

, acute renal D18

nausea, vomiting failure, discharged after

 days

2009 Body aches N/A D15 NONE 2009 Sore throat, N/A D9

nausea, diarrhea 2009 Sore throat, vomiting, N/A D9

headache, confusion 2016 Sore throat N/A D7 NONE 2016

Dehydration, fainting, ER D7 NONE 201

Fatigue, runny nose, Outpatient D7 NONE headache, nausea, vomiting 201

Sore throat, runny N/A D7 NONE nose, tiredness, headache, body aches,

2016 Sore throat ER D11

fatigue, chills 2016 Body ache, nausea ER and Hospitalized fordehydration D8

and difficulty breathing 2014 Sore throat Asthma exacerbation, ER D7

2014 Runny nose Acute COPD exacerbation, ER D7

2014 Runny nose Asthma exacerbation ER D7

2017 Sore throat N/A D15, D

3 NONE 2017 Body ache, runny nose N/A D7, D21 NONE *Initial Symptoms:Fever and cough experienced by all patients; S: seasonal; Pan: pandemic;ER: presented to emergency room; COPD: Chronic obstructive pulmonarydisease; OA: Osteoarthritis; CHF: Congestive heart failure.

indicates data missing or illegible when filedInfection-Induced Anti-NA Antibodies Bind Epitopes that are notPreserved in Current Influenza Vaccines

Experiments were conducted during development of embodiments herein todetermine whether the greater induction of NA-reactive plasmablastsduring natural infection compared to vaccination is because the live,replicating virus displays epitopes not present in the inactivatedvaccines. Memory to conserved epitopes appears to play a role in theobserved bias, as serological studies have shown an induction ofNA-reactive antibodies to past strains (Rajendran et al., 2017; hereinincorporated by reference in its entirety). Both HA and NA antibodieswere encoded from highly mutated variable genes, supporting a memorycell recall origin (FIG. 8). Furthermore, primary exposure to the 2009pandemic influenza virus strain induced NA-reactive plasmablasts atdetectable frequencies in only two of the five infected patients that wecharacterized (top row of FIG. 1D). Conversely, exposure to that strainseven years later in 2016 or to H3N2 strains that have circulated since1968 readily induced NA-reactive plasmablasts (FIGS. 1A and 1D). Todetermine if infection or exposure to whole virus particles couldaccount for the increased NA targeting, mice were infected with intactvirions as opposed to split/subunit vaccine. For this, mice wereinfected intranasally with a sublethal dose of live 2009 pandemic H1N1virus (A/Netherlands/602/2009) or immunized intranasally with intactvirions of inactivated H3N2 (A/Switzerland/9715293/2013) influenzavirus, followed by an intranasal boost with the respective virus strains30 days later. ELISPOT assays on whole splenocytes eight days aftersecondary infection or immunization was used to measure the proportionsof HA- and NA-reactive IgG-secreting cells that were activated. Similarto what was observed in infected humans, the frequency of NA-reactivecells was common after exposure to whole virions for both the H1N1 andH3N2 strains (FIGS. 2A and 2B). This observation was not dependent onviral replication as the H3N2 influenza strain was inactivated. Notably,as in human infections with an H3N2 virus, more plasmablasts werespecific to N2 than to H3 (FIG. 2B). Together these experiments suggestthat NA epitopes present on whole virions are not efficiently targetedby current influenza vaccines. To address this possibility directly, theNA- and HA-reactive mAbs generated from infection-induced plasmablastswere tested for binding to inactivated influenza virus vaccines. Thisanalysis was done on vaccines not expired and with matching influenzavirus strains to those causing infection. While HA-reactive mAbs boundrHA protein with equal affinity to the vaccine, the NA-reactive mAbs hadonly negligible binding to the FLUARIX (FIGS. 2C and 2D) or FLUZONE(FIGS. 2E and 2F) vaccines. The FLUBLOK recombinant protein vaccine hasno NA component at all and so also would not induce NA-reactiveantibodies. Experiments conducted during development of embodimentsherein demonstrate that current influenza virus vaccines haveinsufficient NA content or NA protein structural integrity to induceNA-reactive antibody responses efficiently.

Human NA-Reactive mAbs are More Broadly Reactive than HA-Reactive mAbs

To determine the breadth of binding of the NA-reactive mAbs induced byinfection, ELISA was used to test binding against a diverse panel of rNAproteins (FIG. 3A). All of the N2-reactive mAbs were cross-reacted toall contemporary H3N2 influenza strains, and also a surprising 86% (12of 14) reacted to the first pandemic H3N2 virus strain known to infecthumans (A/Hong Kong/1/1968). Also, 71% (10 of 14) of the antibodiesreacted to the H2N2 influenza strain from 1957 that had circulated inhumans for the eleven years before the H3N2 strain arose. By comparison,only 40% of infection-induced H3-reactive mAbs were cross-reactive tothis 1968 H3N2 strain (FIG. 3B). Similarly, only half of H3-reactivemAbs induced by vaccination in recent years bound to the 1968 H3N2strain (FIG. 3B). Moreover, 64% (9 of 14) of the N2-reactive mAbsinduced by infection were able to bind to avian N2 proteins, includingtwo mAbs with cross-reactivity to heterosubtypic subtypes (N3 and N9)(FIG. 3A). The 2009 pandemic H1N1 influenza strain induced antibodies toHA that were particularly cross-reactive (Li et al., 2012; Wrammert etal., 2011; herein incorporated by reference in their entireties).Analysis conducted during development of embodiments herein demonstratesthat this is also true for N1-reactive mAbs to this strain; 67% of mAbscross-reacted to the 1918 pandemic H1N1 strain, 33% reacted to varioushuman H1N1 strains spanning the entire century, plus 20% bound toheterosubtypic strains (FIG. 3A). Additionally, escape mutants weregenerated to select N2-reactive mAbs that demonstrated broad NIactivity. However, incubating H3N2 (A/Switzerland/9715293/2013) with mAbconcentrations up to 250 μg did not generate escape mutants after manypassages, even though escape mutants arose from highly conservedHA-stalk mAbs (Anderson et al., 2017; herein incorporated by referencein its entirety). This analysis indicates that the epitopes on NA arehighly durable and unlikely to mediate escape by antigenic drift. On thewhole, the NA reactive mAbs induced during influenza virus infectionsare significantly more broadly reactive than antibodies against HA.

NA-Reactive mAbs Show Broad Enzymatic Inhibition Activity In Vitro

The enzymatic function of NA is to cleave the terminal sialic acidresidues allowing viral egress from infected cells. To better access theprotective capacity of the NA-reactive mAbs, inhibition of sialic acidcleavage was evaluated using ELLA and NA-STAR assays. ELLA uses theglycoprotein fetuin as a substrate, detecting mAb-mediated inhibition ofthe sialidase function of NA by any mechanism. These mechanisms includeantibody binding near the enzymatic site or through stericallypreventing interactions between NA and sialic acid residues on fetuinwhen bound more distally from the enzymatic site. Conversely, theNA-Star assay uses a small, soluble chemiluminescent substrate, and somore explicitly distinguishes antibodies that directly inhibit theenzymatic activity of NA by binding near the enzymatic site. Using ELLA,79% (11 of 14) of the N2-reactive mAbs inhibited NA activity against anH3N2 virus, of which about half (5 of 14) were also positive in theNA-STAR assay, demonstrating activity through blockage of the enzymaticdomain directly. By either assay, all of these mAbs inhibited the firstpandemic H3N2 strain A/Hong Kong/1/1968 (FIG. 4A). Therefore, these mAbshave broad NI activity spanning five decades of H3N2 virus evolution.For mAbs reactive to the 2009 pandemic H1N1 strain, 53% (8 of 15) had NIactivity by any means as detected by ELLA, and 20% blocked the enzymaticdomain, showing inhibition via the NA-STAR assay. As with theN2-reactive mAbs, N1-reactive mAbs had broad activity against the 1918pandemic strain A/Brevig Mission/1/1918 (FIG. 4B). These studiesdemonstrate that the majority of human antibodies against NA inhibit theenzymatic activity of this protein on highly divergent influenzastrains.

NA-Reactive Human Monoclonal and Long-Term Polyclonal Antibodies haveHigh Neutralization Activity In Vitro

Microneutralization (MN) measures the inhibition of influenza virusreplication in vitro, providing another correlate of protection. Intotal, 45% of the NA reactive mAbs were able to neutralize virusesrelated to the infecting strain, including; 43% (6 of 14) of theN2-reactive mAbs and 47% (7 of 15) of the N1-reactive mAbs (FIG. 4C). Toensure that the anti-NA antibody response was contributing to long-termserum immunity, NA-reactive polyclonal antibodies were isolated byaffinity purification from two of the patients and tested them by usingMN assays (Lee et al., 2016; herein incorporated by reference in itsentirety). One serum sample was collected at the predicted peak of theimmune response (day 21 post-infection), and the other was obtained wellafter the patient was convalescent at two months (day 63post-infection). The isolated NA-reactive polyclonal antibodies alsoreadily protected MDCK cells from infection in vitro (FIG. 4D). Thesedata show that NA-reactive antibodies commonly exhibit neutralizationactivity, inhibiting virus replication, and contribute to long-termserum immunity.

Identification of NA Residues Crucial for mAb Binding

To map the epitopes recognized by the N1-reactive mAbs, 26 single aminoacid mutant NA proteins from the 2009 pandemic influenza strain wereexpressed in HEK293 cells (Wan et al., 2015; herein incorporated byreference in its entirety). Cell-based ELISAs were carried out to testthe binding of the N1-reactive mAbs to the mutant proteins. A G249Kmutation significantly affected the binding of 1000-3B06 (70% decreasecompared to the wild-type N1). The N273D mutation reduced the binding of1000-1D05 compared to the wild-type N1 protein. Furthermore, the N309Smutation affected both 294-A-1C02 and 294-A-1D05 binding (FIG. 5A).Amino acids N273 and N309 are 99.7% (6835 of 6855 H1 influenza strains)conserved in H1N1 viruses isolated from 1918 until now in the UnitedStates. The G249 site is also conserved in H1N1 viruses (90.3%, 6196 of6855 H1 influenza strains). These residues are all located on the NAhead (FIG. 5B). To map the epitope(s) targeted by the N2-reactive mAbs,ELISA was used to test the binding affinity of N2-reactive mAbs to 12single amino acid mutants of N2 expressed on an A/Minnesota/11/2010(H6N2-PR8 backbone) purified virus. Three amino acids (N221, G248, andG429) on the NA enzymatic conserved domain are critical for the bindingof 229-1D05, 235-1C02 and 235-1E06 (FIGS. 5C and 5D). Consistently, allthree of these mAbs were also positive in the NA-STAR assay (FIG. 4A).These results show that NA-reactive mAbs are readily induced againsthighly conserved epitopes on NA and so are excellent targets forvaccines as well as making the mAbs attractive potential therapeutics.

NA-Reactive mAbs Protect Mice Against Divergent Influenza Viruses

The broad cross-reactivity, as well as widespread in vitro NI activityof NA-reactive mAbs, indicates that they are broadly protective in vivo.The prophylactic protection against challenge was measured withdivergent strains in vivo. Half-maximal lethal dosages (LD50) of theinfluenza virus were determined. Mice received 5 mg/kg of NA-reactivemAb or the same dose of a non-binding control mAb by intraperitonealinjection (i.p.). Two hours later, the mice were lethally challengedwith 10 LD50 of influenza virus by intranasal inoculation. Recent H3N2isolates do not replicate well in the mouse model but historical strainslike A/Philippines/2/1982 (H3N2, X-79) infect mice readily. This virusis phylogenetically distant from recent influenza virus strains,including those that cause the human infections from which the mAbs arederived. Thus, this virus also provides an opportunity to measure thebreadth of protection for the N2-reactive mAbs in vivo. A selection ofN2-reactive mAbs representing all overlapping epitopes were tested. 84%(11 of 13) of the N2-reactive mAbs showed partial or full protection inthe prophylactic challenge experiment against this 35-year-old H3N2influenza strain (FIG. 6A). The protection conferred was consistent withthe breadth of binding and NI activity of these mAbs. Moreover,non-neutralizing NA-reactive mAbs also provided in vivo prophylacticprotection. These data show that neutralizing and non-neutralizingN2-reactive mAbs provide broad prophylactic protection against H3N2influenza strains in vivo.

The larger panel of group 1 influenza strains available for murinechallenge studies allowed a more in-depth analysis of the breadth ofprotection of NA-reactive mAbs. First, mice treated with N1-reactivemAbs were challenged with a 2009 pandemic H1N1 isolate(A/Netherlands/602/2009). Five out of eight of the mAbs from the2009-2010 cohort completely protected mice against weight loss andmortality after challenge, whereas mice treated with control mAb lostweight rapidly and were euthanized by day eight post-infection (FIG.6B). Four out of five of the mAbs that prophylactically protectedagainst H1N1 infection (4 of 8 in total) also provided 100% protectionfrom a highly divergent avian influenza virus strain(A/Vietnam/1203/2004, H5N1) (FIG. 6C). Thus, half of all mAbs inducedagainst N1 in individuals infected with the 2009 pandemic H1N1 strainprovided broad protection against an H5N1 strain. This frequency was farexceeding the 10% of HA-reactive mAbs that arose against this H1N1strain that even bound to H5 (Li et al., 2012; Wrammert et al., 2011;herein incorporated by reference in their entireties). Together, theseresults indicate that when induced against common infectious influenzavirus strains, NA-reactive mAbs are outstanding mediators of broadlyprotective immunity, even to divergent avian influenza virus strainswith pandemic potential.

NA-Reactive mAbs are Excellent Alternatives for Influenza Treatment orProphylaxis

NA inhibitors such as oseltamivir have become the standard of care fortreating influenza virus infections as they have proven efficacy forimproving the outcome of disease (Genentech, 2016; herein incorporatedby reference in its entirety). However, these drugs suffer from dramaticloss of effectiveness if not administered within the first 48 hours ofinfection. Furthermore, the evolution of resistant influenza strains isnow common, severely limiting the usefulness of these drugs. NA-reactivemAbs may be improved alternatives as therapeutic NA-inhibitors, or evenmore efficacious when efficiently elicited by vaccination. As theNA-inhibition antibodies identified had activity against a wide spectrumof influenza virus strains, we tested the activity of these mAbscompared to oseltamivir. Using bio-layer interferometry, an assay wasdevised to competitively measure the binding of oseltamivir versusNA-reactive mAbs to the NA protein. Binding of three of the enzymaticdomain-targeting mAbs (NA-STAR assay positive, 229-1D05, 229-1F06, and229-1G03) is inhibited by prior saturation of NA of anoseltamivir-sensitive strain with oseltamivir (FIGS. 7A and 9). Thisinhibition demonstrates that the binding footprint of the mAbs overlapsat least to some degree with the binding pocket occupied by oseltamivir.Oseltamivir acts by blocking the enzymatic domain, allowing its activityagainst a particular influenza virus strain to be assessed by theNA-STAR assay. While oseltamivir had virtually no NI activity on atypical oseltamivir-resistant strain (A/Texas/12/2007 E119V), all fiveof the enzymatic domain-binding mAbs isolated in this study, which is36% of the N2-reactive mAbs isolated, inhibited the NA activity of thisresistant strain. For 229-1G03 and 235-1E06, the IC50 is nearlyidentical against the sensitive and resistant strains (FIG. 7B).

Additionally, the therapeutic efficacy of the NA-reactive mAbs that wereprotective as prophylactics were also analyzed directly. Mice that werelethally infected with 10 LD50 of influenza virus were treated with 10mg/kg of NA-reactive mAbs 48 hours post-infection. All four of theN1-reactive mAbs fully rescued infected mice from severe weight loss andmortality after 2009 pandemic H1N1 influenza virus challenge (FIG. 7C).Similarly, 88% (7 of 8) of the N2-reactive mAbs proffered full recoveryto the mice challenged with an H3N2 virus (FIG. 7D). In sharp contrast,all mice in the control mAb group had to be euthanized around day ninepost-infection because of severe weight loss. These results show thatthe NA-reactive mAbs are useful therapeutically, even after 48 hours ofinfluenza virus infection, indicating they are alternatives to NAinhibitors such as oseltamivir. With improved vaccine formulations toinduce NA antibodies the same benefits as NA-inhibiting drugs areprophylactically elicited without the need for early administration.Further, unlike NA-inhibiting medications, which lose effectiveness dueto the emergence of resistant strains, administration of boostervaccines would control viral resistance.

All publications and patents provided herein are incorporated byreference in their entireties. Various modifications and variations ofthe described compositions and methods of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thepresent invention.

SEQUENCES The following antibody chain and CDR sequences arereferenced throughout the specification and claimsby their corresponding SEQ ID NOS. and/or names. 228-14-035-2D04 Heavy:(SEQ ID NO: 1) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTAAGCCTGGACAATCGCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAATGCCTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATCAAAACCAAAACTGAAGGCGAGACAGTAGACTACGCTGCACCCGTGAAAGGCAGAATCACCATCTCAAGAGATGACTCAAAGAACATGGTGTATCTGCAATTGAAGAGCCTGAAAATCGAGGACGCAGCCGTTTACTACTGTACCACAGGTCTTACACGTTCGAGTCTCGGCGGCTTCGTTGACTACTGGGGCCCGGGAACCCTGGTCACCGTCTCCTCAGC (SEQ ID NO: 2)EVQLVESGGGLVKPGQSLRLSCAASGFTFTNAWMSWVRQAPGKGLEWVGRIKTKTEGETVDYAAPVKGRITISRDDSKNMVYLQLKSLKIEDAAVYYCTTGLTRSSLGGFVDYWGPGTLVTVSS  CDRH1: (SEQ ID NO: 3) AATGCCTGGATGAGT(SEQ ID NO: 4) NAWMS CDRH2: (SEQ ID NO: 5)CGTATCAAAACCAAAACTGAAGGCGAGACAGTAGACTACGCTGCACCCGT GAAAGGC(SEQ ID NO: 6) RIKTKTEGETVDYAAPVKG CDRH3: (SEQ ID NO: 7)ACCACAGGTCTTACACGTTCGAGTCTCGGCGGCTTCGTTGACTAC (SEQ ID NO: 8)TTGLTRSSLGGFVDY Kappa: (SEQ ID NO: 9)GACATCGTGATGACCCAGTCTCCGGACTCCCTGACTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGGTCCAGCCAGACTGTTTTGTCCAGCTCCAACAATGAGAACTTCTTAGCTTGGTACCAGCAGAAATCAGGACAGCCTCCTAACCTGCTCATTTACTGGGCATCTACCCGGGCATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTATTACTGTCTCCAATATCTTACTACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 10)DIVMTQSPDSLTVSLGERATINCRSSQTVLSSSNNENFLAWYQQKSGQPPNLLIYWASTRASGVPDRFSGSGSGTDFTLTISSLQTEDVAVYYCLQYLTT PRTFGQGTKVEIK CDRK1:(SEQ ID NO: 11) AGGTCCAGCCAGACTGTTTTGTCCAGCTCCAACAATGAGAACTTCTTAGC T(SEQ ID NO: 12) RSSQTVLSSSNNENFLA CDRK2: (SEQ ID NO: 13)TGGGCATCTACCCGGGCATCC (SEQ ID NO: 14) WASTRAS CDRK3: (SEQ ID NO: 15)CTCCAATATCTTACTACTCCTCGGACG (SEQ ID NO: 16) LQYLTTPRT 229-14-036-1D05Heavy: (SEQ ID NO: 17)GTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTAGTAGTAGTACTTACACAGACTACGCAGACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCATTGTATCTACAAATGAACAACCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGACCGTGGCCGACACCGCGTATAGCAGAGGCAGGCCACAAATTACCCACTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGC (SEQ ID NO: 18)VQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWISYISSSSTYTDYADSVKGRFTVSRDNAKNSLYLQMNNLRAEDTAVYYCATVADTAYSRGRPQITHFDNWGQGTLVTVSS CDRH1: (SEQ ID NO: 19) GACTACTACATGAGC(SEQ ID NO: 20) DYYMS CDRH2: (SEQ ID NO: 21)TACATTAGTAGTAGTAGTACTTACACAGACTACGCAGACTCTGTGAAGGG C (SEQ ID NO: 22)YISSSSTYTDYADSVKG CDRH3: (SEQ ID NO: 23)GCGACCGTGGCCGACACCGCGTATAGCAGAGGCAGGCCACAAATTACCCA CTTTGACAAC(SEQ ID NO: 24) ATVADTAYSRGRPQITHFDN Lambda: (SEQ ID NO: 25)TCCTATGAGCTGACTCAGCCACCCTCAATGTCCGTGTCCCCAGGACAGACAGCCACCATCACCTGTTTTGGAGATAAATTGGGGGAAAAGTATGCTTACTGGTATCAGCAGAAGCCTGGCCAGTCCCCTCTACTGGTCATCTATCAAGATACCAAGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCAGACGTGGGACAGCACCCTTGTGTTTTTCGGCGGAGGG ACCAAGCTGACCGTCCTAG(SEQ ID NO: 26) SYELTQPPSMSVSPGQTATITCFGDKLGEKYAYWYQQKPGQSPLLVIYQDTKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQTWDSTLVFFGGG TKLTVL CDRL1:(SEQ ID NO: 27) TTTGGAGATAAATTGGGGGAAAAGTATGCTTAC (SEQ ID NO: 28)FGDKLGEKYAY CDRL2: (SEQ ID NO: 29) CAAGATACCAAGCGGCCCTCA (SEQ ID NO: 30)QDTKRPS CDRL3: (SEQ ID NO: 31) CAGACGTGGGACAGCACCCTTGTGTTT(SEQ ID NO: 32) QTWDSTLVF 229-14-036-1G03 Heavy: (SEQ ID NO: 33)GTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTAAGACTCTCCTGTGCAGTGTCTGGACTCACCATCAATGACCTTGTCATCCACTGGGTCCGCCAGCCTCCAGACAAGGGGCTGGAGTGGGTGGCAGTTATGGGGTATGATGGCGGAAACAAAGACTATGCAGAATCCGTGAAGGGCCGATTCAGCATCTCCGGGGACAATCCCCAGAACACACTGTATCTGCAGATAAACAGCCTGAGAGTCGAGGACACGGCTGTATATTACTGTGCGAGAGCATCATACTTCGGGGAGTTAAGAGACGAGTACTACTCCTTCGCCATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG (SEQ ID NO: 34)VQLVESGGGVVQPGGSLRLSCAVSGLTINDLVIHWVRQPPDKGLEWVAVMGYDGGNKDYAESVKGRFSISGDNPQNTLYLQINSLRVEDTAVYYCARASYFGELRDEYYSFAMDVWGQGTTVTVSS CDRH1: (SEQ ID NO: 35) GACCTTGTCATCCAC(SEQ ID NO: 36) DLVIH CDRH2: (SEQ ID NO: 37)GTTATGGGGTATGATGGCGGAAACAAAGACTATGCAGAATCCGTGAAGGG C (SEQ ID NO: 38)VMGYDGGNKDYAESVKG CDRH3: (SEQ ID NO: 39)GCGAGAGCATCATACTTCGGGGAGTTAAGAGACGAGTACTACTCCTTCGC CATGGACGTC(SEQ ID NO: 40) ARASYFGELRDEYYSFAMDV Kappa: (SEQ ID NO: 41)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGGCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGTAGGAGTTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCACTGTATTACTGTCAGCTGTATGGTACCTCACCTCCGTACACTTTTGGCCAGGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 42)EIVLTQSPGTLSLSPGERGTLSCRASQSVSRSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFALYYCQLYGTSPPYTF GQGTKVEIK CDRK1:(SEQ ID NO: 43) AGGGCCAGTCAGAGTGTTAGTAGGAGTTACTTAGCC (SEQ ID NO: 44)RASQSVSRSYLA CDRK2: (SEQ ID NO: 45) GGTGCATCCAGCAGGGCCACT(SEQ ID NO: 46) GASSRAT CDRK3: (SEQ ID NO: 47)CAGCTGTATGGTACCTCACCTCCGTACACT (SEQ ID NO: 48) QLYGTSPPYT229-14-036-2B04 Heavy: (SEQ ID NO: 49)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTGTCAGTAATGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTTGGTCGTATTAAGAAAGAAAGTGAGGGTGGGACAATAGACTACGGTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGAATCAAAAAACATATTGTATCTGCACATGAAGAGCCTGATAACCGATGACACAGCCGTGTACTACTGTACCATCCCGAATCCTCAAATTGTGGTGGTGACTACTACTCCACATTCCCATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGC (SEQ ID NO: 50)EVQLVESGGGLVKPGGSLRLSCAASGFTVSNAWMSWVRQAPGKGLEWVGRIKKESEGGTIDYGAPVKGRFTISRDESKNILYLHMKSLITDDTAVYYCTIPNPQIVVVTTTPHSHWGQGTLVTVSS CDRH1: (SEQ ID NO: 51) AATGCCTGGATGAGC(SEQ ID NO: 52) NAWMS CDRH2: (SEQ ID NO: 53)CGTATTAAGAAAGAAAGTGAGGGTGGGACAATAGACTACGGTGCACCCGT GAAAGGC(SEQ ID NO: 54) RIKKESEGGTIDYGAPVKG CDRH3: (SEQ ID NO: 55)ACCATCCCGAATCCTCAAATTGTGGTGGTGACTACTACTCCACATTCCCA T (SEQ ID NO: 56)TIPNPQIVVVTTTPHSH Lambda: (SEQ ID NO: 57)TCCTATGAGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAATGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGTTGGTCATCTATTATGATAGTGACCGGCCCTCAGCGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTG----TTCGGCGGAGGGACCAAGCTGGCCGTCCTAG (SEQ ID NO: 58)SYELTQPPSVSVAPGKTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYYDSDRPSAIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFG GGTKLAVL CDRL1:(SEQ ID NO: 59) GGGGGAAACAACATTGGAAGTAAAAATGTGCAC (SEQ ID NO: 60)GGNNIGSKNVH CDRL2: (SEQ ID NO: 61) TATGATAGTGACCGGCCCTCA (SEQ ID NO: 62)YDSDRPS CDRL3: (SEQ ID NO: 63) CAGGTGTGGGATAGTAGTAGTGATCATTGGGTG(SEQ ID NO: 64) QVWDSSSDHWV 229-14-036-2C06 Heavy: (SEQ ID NO: 65)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTCGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTTAAAAACTTCGCCATGACCTGGGTCCGCCTGTCTCCAGGGAAGGGACTGGAGTGGGTCTCATCCATAAGCGGAGACGGTGGAAGGACCTACTACTCAGAATCTGCTAAGGGACGGTTAATCATCTCCAGAGACAATGCCAACAACAGGCTGTTTCTACAAATGTACAGCCTGAGAGCCGACGACACGGCCATATATTTCTGTGCGAAAGATCGGGTGTCGCTGTGGTTCGGGGAGAACAGGGGCTGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGC (SEQ ID NO: 66)EVQLLESGGGSVQPGGSLRLSCEASGFTFKNFAMTWVRLSPGKGLEWVSSISGDGGRTYYSESAKGRLIISRDNANNRLFLQMYSLRADDTAIYFCAKDRVSLWFGENRGWFDSWGQGTLVTVSS CDRH1: (SEQ ID NO: 67) AACTTCGCCATGACC(SEQ ID NO: 68) NFAMT CDRH2: (SEQ ID NO: 69)TCCATAAGCGGAGACGGTGGAAGGACCTACTACTCAGAATCTGCTAAGGG A (SEQ ID NO: 70)SISGDGGRTYYSESAKG CDRH3: (SEQ ID NO: 71)GCGAAAGATCGGGTGTCGCTGTGGTTCGGGGAGAACAGGGGCTGGTTCGA CTCC (SEQ ID NO: 72)AKDRVSLWFGENRGWFDS Lambda: (SEQ ID NO: 73)AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTGACCATCTCCTGCACCGGCAGCAGTGGCAACATCGCCCGCTTCTCTGTGCAGTGGTATCAGCAACGCCCGGGCAGTGGCCCTATCACTGTGATCTATGAGAATAGTCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACACCTCCTCCAATTCTGCCTCCCTCACCATCTCTGGACTGAAGATTGAAGACGAGGGAGACTACTACTGTCAGTCTTATGATCTCAACAATTATTGGGTGTTCGGCGGAGGGACCAAACTGACCGTCCTA (SEQ ID NO: 74)NFMLTQPHSVSESPGKTVTISCTGSSGNIARFSVQWYQQRPGSGPITVIYENSQRPSGVPDRFSGSIDTSSNSASLTISGLKIEDEGDYYCQSYDLNNYW VFGGGTKLTVL CDRL1:(SEQ ID NO: 75) ACCGGCAGCAGTGGCAACATCGCCCGCTTCTCTGTGCAG (SEQ ID NO: 76)TGSSGNIARFSVQ CDRL2: (SEQ ID NO: 77) GAGAATAGTCAAAGACCCTCT(SEQ ID NO: 78) ENSQRPS CDRL3: (SEQ ID NO: 79)CAGTCTTATGATCTCAACAATTATTGGGTG (SEQ ID NO: 80) QSYDLNNYWV235-15-042-1E06 Heavy: (SEQ ID NO: 81)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCATCTTCAGAAGTTATGAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGATTTCATACATTAGTAGTAGTGGTTCAACCATGTTCTACGCAGACTCTGTGAAGGGCCGATTCACCGTCTCCAGAGGCAATGGCGAGAACTCACTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAAATGGCCCAAAAGAAGGCAGCAGTTGGGACGACTGGTTCGACCCCTGGGGCCAGGGAACTCTGGTCACCGTCTCCTCAGC (SEQ ID NO: 82)EVQLVESGGGLVQPGGSLRLSCAASGFIFRSYEMNWVRQAPGKGLEWISYISSSGSTMFYADSVKGRFTVSRGNGENSLYLQMDSLRAEDTAVYYCARNGPKEGSSWDDWFDPWGQGTLVTVSS CDRH1: (SEQ ID NO: 83) AGTTATGAAATGAAC(SEQ ID NO: 84) SYEMN CDRH2: (SEQ ID NO: 85)TACATTAGTAGTAGTGGTTCAACCATGTTCTACGCAGACTCTGTGAAGGG C (SEQ ID NO: 86)YISSSGSTMFYADSVKG CDRH3 : (SEQ ID NO: 87)GCGAGAAATGGCCCAAAAGAAGGCAGCAGTTGGGACGACTGGTTCGACCC C (SEQ ID NO: 88)ARNGPKEGSSWDDWFDP Lambda: (SEQ ID NO: 89)TCCTATGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAATCAGGATCACATGCCAAGGAGACACCCTCAGAAGCTATTCTGCAAGTTGGTACCAGCAGAAGCCAGGACAGGCCCCTCTAGTTGTCATCTTTGGTGATAACAATAGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGGTTAGGAGACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAGTTCCCGGGACAGCAATAACAACCCCCTATATGTCTTCGGAACTGGGACCAAGGTCACCGTCC (SEQ ID NO: 90)SYELTQDPAVSVALGQTIRITCQGDTLRSYSASWYQQKPGQAPLVVIFGDNNRPSGIPDRFSGSRLGDTASLTITGAQAEDEADYYCSSRDSNNNPLYVF GTGTKVTV CDRL1:(SEQ ID NO: 91) CAAGGAGACACCCTCAGAAGCTATTCTGCAAGT (SEQ ID NO: 92)QGDTLRSYSAS CDRL2: (SEQ ID NO: 93) GGTGATAACAATAGGCCCTCA (SEQ ID NO: 94)GDNNRPS CDRL3 : (SEQ ID NO: 95) AGTTCCCGGGACAGCAATAACAACCCCCTATATGTC(SEQ ID NO: 96) SSRDSNNNPLYV 1000-2E06 Heavy: (SEQ ID NO: 97)GTGCAGCTGGTGCAGTCTGGGCCTGAGGTGAAGAAGTCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGATACACCTTCAGTAACTATGCTGTACATTGGGTGCGCCAGGCCCCCGGACAAAGGCCTGAGTGGATGGGGTGGAGCAACGCTGGCAGTGGTGCCACAAAATATTCACAGAATTTCCAGGGCAGACTCACCATTGTCAGGGACACATCCGCGAACACAGTCTTCATGGAGCTGAGCAGCCTGACATCTGAGGACACGGCTGTATATTACTGTGCGAGACCAGTGAGAAACGGCATAGCACCTAGTGCTATCGAATACTGGGGCCAGGGAACCCTGGT CACCGTCTCCTCAGC(SEQ ID NO: 98) VQLVQSGPEVKKSGASVKISCKASGYTFSNYAVHWVRQAPGQRPEWMGWSNAGSGATKYSQNFQGRLTIVRDTSANTVFMELSSLTSEDTAVYYCARPVR NGIAPSAIEYWGQGTLVTVSSCDRH1: (SEQ ID NO: 99) AACTATGCTGTACAT (SEQ ID NO: 100) NYAVH CDRH2:(SEQ ID NO: 101) TGGAGCAACGCTGGCAGTGGTGCCACAAAATATTCACAGAATTTCCAGGG C(SEQ ID NO: 102) WSNAGSGATKYSQNFQG CDRH3: (SEQ ID NO: 103)GCGAGACCAGTGAGAAACGGCATAGCACCTAGTGCTATCGAATAC (SEQ ID NO: 104)ARPVRNGIAPSAIEY Kappa: (SEQ ID NO: 105)GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTTTACAGGTCCACCAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTCACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAATACGATCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC (SEQ ID NO: 106)DIVMTQSPDSLAVSLGERATINCKSSQSVFYRSTNKNYLAWYQQKPGQPPKLLIHWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNT ITFGPGTKVDIK CDRK1:(SEQ ID NO: 107) AAGTCCAGCCAGAGTGTTTTTTACAGGTCCACCAATAAGAACTACTTAGC T(SEQ ID NO: 108) KSSQSVFYRSTNKNYLA CDRK2: (SEQ ID NO: 109)TGGGCATCTACCCGGGAATCC (SEQ ID NO: 110) WASTRES CDRK3: (SEQ ID NO: 111)CAGCAATATTATAATACGATCACT (SEQ ID NO: 112) QQYYNTIT 294-16-009-A-1C02Heavy: (SEQ ID NO: 113)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCCTCAGTTGTGGTACTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGATCTGGAGTGGCTTGGGAGTATCTATTGTAGTGGAAACACCTACTACAACCCGTCCCTCAAGAGTCAAGTCACCATATCCGTGGACACGTCCAAGAAAGAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACATGCAGGACATCTTGCGCCTTTTGGAGTGGACCTAACTGATGGTTTTGATATCTGGGGCCGAGGGACAATGGTCACCGTCTCTTCAGC (SEQ ID NO: 114)QVQLQESGPGLVKPSETLSLTCTVSGGSLSCGTYYWGWIRQPPGKDLEWLGSIYCSGNTYYNPSLKSQVTISVDTSKKEFSLKLSSVTAADTAVYYCARHAGHLAPFGVDLTDGFDIWGRGTMVTVSS CDRH1: (SEQ ID NO: 115)TGTGGTACTTACTACTGGGGC (SEQ ID NO: 116) CGTYYWG CDRH2: (SEQ ID NO: 117)AGTATCTATTGTAGTGGAAACACCTACTACAACCCGTCCCTCAAGAGT (SEQ ID NO: 118)SIYCSGNTYYNPSLKS CDRH3: (SEQ ID NO: 119)GCGAGACATGCAGGACATCTTGCGCCTTTTGGAGTGGACCTAACTGATGG TTTTGATATC(SEQ ID NO: 120) ARHAGHLAPFGVDLTDGFDI Lambda: (SEQ ID NO: 121)CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCCGGGGCCCCAGGACAGAGGGTCACCATCTCCTGCACTGGGAGTAGTTCCAACATTGGGGCAGGTTATGATGTACACTGGTATCAGAAGCTTCCAGCAACAGCCCCCAAACTCCTCATCTATGGTAACAACAATCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAACAGCCTGAGTGGTTTTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQKLPATAPKLLIYGNNNRPSGV (SEQ ID NO: 122)PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDNSLSGFVVFGGGTKLT V CDRL1:(SEQ ID NO: 123) ACTGGGAGTAGTTCCAACATTGGGGCAGGTTATGATGTACAC(SEQ ID NO: 124) TGSSSNIGAGYDVH CDRL2: (SEQ ID NO: 125)GGTAACAACAATCGACCCTCA (SEQ ID NO: 126) GNNNRPS CDRL3: (SEQ ID NO: 127)CAGTCCTATGACAACAGCCTGAGTGGTTTTGTGGTA (SEQ ID NO: 128) QSYDNSLSGFVV294-16-009-A-1C06 Heavy: (SEQ ID NO: 129)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAGGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCCTGGCTATAGCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAATGGTAATAGTAATTCCATATACTACGGAGACTCAGTGAAGGGCCGGTTCACCATCGCCAGAGACAACGCCAAGAACTTACTATATCTGCAAATGAACAGCCTGAGGGCCGACGACACGGCTATTTATTACTGTGCGAGAGGCGGCGTAGCACTGGCTCAGGCTGACTACTGGGGCCAGGGAGCCCTGGTCACCGT CTCCTCAGC(SEQ ID NO: 130) EVQLVESGGGLVRPGGSLRLSCAASGFTFPGYSMSWIRQAPGKGLEWVSSINGNSNSIYYGDSVKGRFTIARDNAKNLLYLQMNSLRADDTAIYYCARGG VALAQADYWGQGALVTVSSCDRH1: (SEQ ID NO: 131) GGCTATAGCATGAGC (SEQ ID NO: 132) GYSMS CDRH2:(SEQ ID NO: 133) TCCATTAATGGTAATAGTAATTCCATATACTACGGAGACTCAGTGAAGGG C(SEQ ID NO: 134) SINGNSNSIYYGDSVKG CDRH3: (SEQ ID NO: 135)GCGAGAGGCGGCGTAGCACTGGCTCAGGCTGACTAC (SEQ ID NO: 136) ARGGVALAQADYKappa: (SEQ ID NO: 137)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTACCACCTTGTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCGCTGCTGCATCCAGTTTGCAAAGGGGGGTCCCATCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCATGAGTCTGCAACCTGAAGATGTTGCGACTTACTACTGTCACCAGACTTACAAAACCTTGTGGACGTTCGGCCAGGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 138)DIQMTQSPSSLSASVGDRVTITCRASQSITTLLNWYQQKPGKAPKLLIAAASSLQRGVPSRFSGSGSGTDFTLTIMSLQPEDVATYYCHQTYKTLWTFGQ GTKVEIK CDRK1:(SEQ ID NO: 139) CGGGCAAGTCAGAGCATTACCACCTTGTTAAAT (SEQ ID NO: 140)RASQSITTLLN CDRK2: (SEQ ID NO: 141) GCTGCATCCAGTTTGCAAAGG(SEQ ID NO: 142) AASSLQR CDRK3: (SEQ ID NO: 143)CACCAGACTTACAAAACCTTGTGGACG (SEQ ID NO: 144) HQTYKTLWT 294-16-009-A-1D05Heavy: (SEQ ID NO: 145)CAGGTGCAGCTGCAGGAGTCCGACTCAGGACTGGTCAGGCCCTCACAGACCCTGTCACTCACCTGCGCTGTCTCTGGTGACTCCATCACCACTAGCACTTACTCCTGGAATTGGATCCGGCAGACACCAGGGAAGGGCCTGGAGTGGATTGGATATATCTATCCTGCTGGGAGTCCCATCTACAATCCGTCCCTGAAGGGTCGAGTCACTATATCAATAGACAAGTCCAAAAACCAGTTCTCCCTGAACTTGAGCTCTGTGACCGCCGCGGACACGGCCATGTATTACTGTGCCACCCGGTCTAGACCGACAATTGGTATTGGTGCTTACGATGTCTGGGGCCAAGGGAC AATGGTCACCGTCTCTTCAGC(SEQ ID NO: 146) QVQLQESDSGLVRPSQTLSLTCAVSGDSITTSTYSWNWIRQTPGKGLEWIGYIYPAGSPIYNPSLKGRVTISIDKSKNQFSLNLSSVTAADTAMYYCATRSRPTIGIGAYDVWGQGTMVTVSS CDRH1: (SEQ ID NO: 147) ACTAGCACTTACTCCTGGAAT(SEQ ID NO: 148) TSTYSWN CDRH2: (SEQ ID NO: 149)TATATCTATCCTGCTGGGAGTCCCATCTACAATCCGTCCCTGAAGGGT (SEQ ID NO: 150)YIYPAGSPIYNPSLKG CDRH3: (SEQ ID NO: 151)GCCACCCGGTCTAGACCGACAATTGGTATTGGTGCTTACGATGTC (SEQ ID NO: 152)ATRSRPTIGIGAYDV Kappa: (SEQ ID NO: 153)GAAATAGTGATGACGCAGTCTCCAGCCGCCCTGTCTGTGTCTCTAGGGGGTAGAGCCACCCTCTCCTGCAGGGCCACTGAGCGTGTTAACAGCGACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGTGCATCCACCAGGGCCTCTAATGTCCCAGCCAGGTTCAGTGGCGGTGGGTCTGGAACAGACTTCATTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGGAGTTTACTACTGTCAGCAGTATAAGACCTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 154)EIVMTQSPAALSVSLGGRATLSCRATERVNSDLAWYQQKPGQAPRLLIYGASTRASNVPARFSGGGSGTDFILTISSLQSEDFGVYYCQQYKTWPRTFGQ GTKVEIK CDRK1:(SEQ ID NO: 155) AGGGCCACTGAGCGTGTTAACAGCGACTTAGCC (SEQ ID NO: 156)RATERVNSDLA CDRK2: (SEQ ID NO: 157) GGTGCATCCACCAGGGCCTCT(SEQ ID NO: 158) GASTRAS CDRK3: (SEQ ID NO: 159)CAGCAGTATAAGACCTGGCCTCGGACG (SEQ ID NO: 160) QQYKTWPRT 294-16-009-G-1F01Heavy: (SEQ ID NO: 161)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTACGAGCTATTGGATGAGCTGGGTCCGCCAGACTCCAGGGAAAGGGCTGGAGTGGGTGGCCAACATAAAGGAAGATGGAAGTCAGAAATACCATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTATTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGCTCATGAGTCGTTCTATTTCTCTGGTAGTACTACTTTTTACGCCGGACCGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGC (SEQ ID NO: 162)EVQLVESGGGLVQPGGSLRLSCAVSGFTFTSYWMSWVRQTPGKGLEWVANIKEDGSQKYHVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARAHESFYFSGSTTFYAGPGAFDIWGQGTMVTVSS CDRH1: (SEQ ID NO: 163) AGCTATTGGATGAGC(SEQ ID NO: 164) SYWMS CDRH2: (SEQ ID NO: 165)AACATAAAGGAAGATGGAAGTCAGAAATACCATGTGGACTCTGTGAAGGG C (SEQ ID NO: 166)NIKEDGSQKYHVDSVKG CDRH3: (SEQ ID NO: 167)GCGAGAGCTCATGAGTCGTTCTATTTCTCTGGTAGTACTACTTTTTACGCCGGACCGGGGGCTTTTGATATC (SEQ ID NO: 168) ARAHESFYFSGSTTFYAGPGAFDI Lambda:(SEQ ID NO: 169) CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATATTGGGAGTTATAAACTTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCCAACTCTTGATTTATGACGTCAGTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTCATATGCAGGTAGTAGCATTGTGCTTTTCGGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO: 170)QSALTQPASVSGSPGQSITISCTGTSSDIGSYKLVSWYQQHPGKAPQLLIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSSIVL FGGGTKLTVL CDRL1:(SEQ ID NO: 171) ACTGGAACCAGCAGTGATATTGGGAGTTATAAACTTGTCTCC(SEQ ID NO: 172) TGTSSDIGSYKLVS CDRL2: (SEQ ID NO: 173)GACGTCAGTAAGCGGCCCTCA (SEQ ID NO: 174) DVSKRPS CDRL3: (SEQ ID NO: 175)TGCTCATATGCAGGTAGTAGCATTGTGCTT (SEQ ID NO: 176) CSYAGSSIVL296-16-003-G-2F04 Heavy: (SEQ ID NO: 177)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACTTGTACCATGAACTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTACTAGTACTTCCATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAACAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCCGGGATAATTGGAAGTACGGCGGACTACTACTACATCGACGTCTGGGGCAAAGGGACCAC GGTCACCGTCTCCTCAG(SEQ ID NO: 178) EVQLVESGGGLVKPGGSLRLSCAASGFTFSTCTMNWVRQVPGKGLEWVSSISSTSTSIYYADSVKGRFTISRDNANNSLYLQMNSLRAEDTAVYYCAGIIGSTADYYYIDVWGKGTTVTVSS CDRH1: (SEQ ID NO: 179) ACTTGTACCATGAAC(SEQ ID NO: 180) TCTMN CDRH2: (SEQ ID NO: 181)TCCATTAGTAGTACTAGTACTTCCATATACTACGCAGACTCAGTGAAGGG C (SEQ ID NO: 182)SISSTSTSIYYADSVKG CDRH3: (SEQ ID NO: 183)GCCGGGATAATTGGAAGTACGGCGGACTACTACTACATCGACGTC (SEQ ID NO: 184)AGIIGSTADYYYIDV Kappa: (SEQ ID NO: 185)GACATCCAGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCTTCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCACCAGCTTAATAGTTACCGCTACACTTTCGGCGGAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 186)DIQMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQLNSYRYTFGG GTKVEIK CDRK1:(SEQ ID NO: 187) CGGGCCAGTCAGGGCATTAGCAGTTATTTAGCC (SEQ ID NO: 188)RASQGISSYLA CDRK2: (SEQ ID NO: 189) GCTGCTTCCACTTTGCAAAGT(SEQ ID NO: 190) AASTLQS CDRK3: (SEQ ID NO: 191)CACCAGCTTAATAGTTACCGCTACACT (SEQ ID NO: 192) HQLNSYRYT 300-16-005-G-2A04Heavy: (SEQ ID NO: 193)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGATTGGTGAAGTCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGCCTCCATCAGCAGTGATTATTACTTCTGGACCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAATGGATTGGGTACATCTATACCAGTGGGAGCAGTAGTTACAATCCCTCCCTCAGGAGTCGAGTCAGCATATCAGTAGACACGTCCAAGAACCACTTCTCCCTGAAGCTGAGCTCTGTGACCGCCACAGACACGGCCGTGTATTACTGTGCGAGAGAAGTGGCACGGGATACCAGTGGTTATTACTACTACTTTGATTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGC (SEQ ID NO: 194)QVQLQESGPGLVKSSQTLSLTCTVSGASISSDYYFWTWIRQPAGKGLEWIGYIYTSGSSSYNPSLRSRVSISVDTSKNHFSLKLSSVTATDTAVYYCAREVARDTSGYYYYFDSWGQGTLVTVSS CDRH1: (SEQ ID NO: 195) AGTGATTATTACTTCTGGACC(SEQ ID NO: 196) SDYYFWT CDRH2: (SEQ ID NO: 197)TACATCTATACCAGTGGGAGCAGTAGTTACAATCCCTCCCTCAGGAGT (SEQ ID NO: 198)YIYTSGSSSYNPSLRS CDRH3: (SEQ ID NO: 199)GCGAGAGAAGTGGCACGGGATACCAGTGGTTATTACTACTACTTTGATTC C (SEQ ID NO: 200)AREVARDTSGYYYYFDS Lambda: (SEQ ID NO: 201)CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGAAGTACACTGGTACCAGCAGTTTCCAGGAACAGCCCCCAAACTCCTCATCTATGCTGACTACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAGGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGACTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAACACTTTGAAACTCTTCGGAACTGGGACCAAGGTCACCGTCCT (SEQ ID NO: 202)QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYEVHWYQQFPGTAPKLLIYADYNRPSGVPDRFSGSRSGTSASLAITGLQAEDEADYYCQSYDNTLKLF GTGTKVTV CDRL1:(SEQ ID NO: 203) ACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGAAGTACAC(SEQ ID NO: 204) TGSSSNIGAGYEVH CDRL2: (SEQ ID NO: 205)GCTGACTACAATCGGCCCTCA (SEQ ID NO: 206) ADYNRPS CDRL3: (SEQ ID NO: 207)CAGTCCTATGACAACACTTTGAAACTC (SEQ ID NO: 208) QSYDNTLKL 229 1D02 Heavy:(SEQ ID NO: 209) EVQLVESGGGLVKPGGSLRLACAASGFSLSNYSMTWVRQAPGKELEWVSSIGSSSNYIEYAGSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCARDF GYEFDFWGQGSLVTVSSCDRH1: (SEQ ID NO: 210) AASGFSLSNYSMT CDRH2: (SEQ ID NO: 211) SIGSSSNYIECDRH3: (SEQ ID NO: 212) ARDFGYEFDF Kappa: (SEQ ID NO: 213)DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTP TFGQGTKVEIK CDRL1:(SEQ ID NO: 214) RSSQSLLYSNGYNYLD CDRL2: (SEQ ID NO: 215) YLGSNRASCDRL3: (SEQ ID NO: 216) MQGLQTPT 229 1F06 Heavy: (SEQ ID NO: 217)VQLVESGGGVVQPGRSLRLSCTSSGFHFNDYFMHWVRQAPGNGLEWVAVMGHDGSNKDFSDSMKGRATISGDNSQNTLYLQINSLRVEDSAVYYCARASYFGELRADHYSFAMDVWGQGTMVTVSS CDRH1: (SEQ ID NO: 218) TSSGFHFNDYFMH CDRH2:(SEQ ID NO: 219) VMGHDGSNKD CDRH3: (SEQ ID NO: 220) ARASYFGELRADHYSFAMDVKappa: (SEQ ID NO: 221)EIVLTQSPGILSLSPGERGTLSCRASQSVSRSDLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYGTSPPYTF GQGTKVEIK CDRL1:(SEQ ID NO: 222) RASQSVSRSDLA CDRL2: (SEQ ID NO: 223) YGASSRAT CDRL3:(SEQ ID NO: 224) QQYGTSPPYT 229 2DO3 Heavy: (SEQ ID NO: 225)EVQLVESGGGLVQPGGSLRLSCAVSGLTVSGNYMSWVRQAPGKGLEWVSVLYTNGKTFYADSVKGRFIISRDNAKNTLSLQMNSLRAEDTAVYFCTTNWD FYYYFNNWGQGTLVTVSSCDRH1: (SEQ ID NO: 226) AVSGLTVSGNYMS CDRH2: (SEQ ID NO: 227) VLYTNGKTFCDRH3: (SEQ ID NO: 228) TTNWDFYYYFNN Kappa: (SEQ ID NO: 229)DIQMTQSPSTLSASVGDRVTITCRASQGITTWLAWYQQKPGKAPRLLIYQASSLESGVPLRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNYPYTFGQ GTKVEIK CDRL1:(SEQ ID NO: 230) RASQGITTWLA CDRL2: (SEQ ID NO: 231) YQASSLES CDRL3:(SEQ ID NO: 232) QQYNNYPYT

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1. A binding agent comprising: (a) a polypeptide comprising a regionhaving at least 70% sequence identity with a polypeptide encoded by oneof SEQ ID NOs. 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, and193; and (b) a polypeptide comprising a region having at least 70%sequence identity with a polypeptide encoded by one of SEQ ID NOs. 9,25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, and 201; wherein thebinding agent exhibits similar influenza epitope-binding characteristicsto an antibody selected from the group consisting of 228-14-035-2D04,229-14-036-1D05, 229-14-036-1G03, 229-14-036-2B04, 229-14-036-2C06,235-15-042-1E06, 1000-2E06, 294-16-009-A-1C02, 294-16-009-A-1C06,294-16-009-A-1D05, 294-16-009-G-1F01, 296-16-003-G-2F04, and/or300-16-005-G-2A04.
 2. The binding agent of claim 1, wherein thepolypeptide of (a) and the polypeptide of (b) comprise first and secondpolypeptides.
 3. The binding agent of claim 2, wherein the binding agentis a monoclonal antibody or monobody.
 4. The binding agent of claim 2wherein the binding agent is an antibody fragment.
 5. The binding agentof claim 1, wherein the polypeptide of (a) and the polypeptide of (b)are a single polypeptide chain.
 6. The binding agent of claim 1,comprising binding affinity for an epitope displayed on two or moredifferent virus strains.
 7. The binding agent of claim 6, wherein thetwo or more different virus strains are influenza strains.
 8. Thebinding agent of claim 7, wherein a first influenza strain and thesecond influenza strains comprise different neuraminidase types.
 9. Thebinding agent of claim 1, wherein the binding agent has neuraminidaseinhibitory activity.
 10. The binding agent of claim 1, wherein thebinding agent lacks neuraminidase inhibitory activity.
 11. The bindingagent of claim 1, wherein the binding agent has an affinity for theepitope of at least 10⁷ M⁻¹.
 12. A polynucleotide comprising a potionwith 70% or greater sequence identity with one or more of SEQ ID NOs: 1,17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 9, 25, 41, 57, 73,89, 105, 121, 137, 153, 169, 185, and
 201. 13. A polynucleotide orpolynucleotides encoding a binding agent of one of claim 1-11.
 14. Apharmaceutical preparation comprising a binding agent of one of claim1-11.
 15. A method comprising administering a therapeutic dose of thepharmaceutical preparation of claim 14 to a subject.
 16. The method ofclaim 15, wherein the subject is human.
 17. The method of claim 15,wherein the subject is infected with influenza.
 18. The method of claim15, wherein the subject is at risk of influenza infection.
 19. Themethod of claim 15, wherein said binding agent is co-administered withone or more additional therapeutic agents.
 20. The method of claim 19,wherein said one or more additional therapeutic agents are selected fromthe group consisting of antivirals, immunologic agents, antibiotics, andagents for relieving symptoms of influenza infection.
 21. A bindingagent that neutralizes infection of one or more strains of influenza Avirus and binds the same epitope as a binding agent comprising: (i) aheavy chain variable region comprising a CDR1 of SEQ ID NO: 4, a CDR2 ofSEQ ID NO: 6 and CDR3 of SEQ ID NO: 8, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 12, a CDR2 of SEQ ID NO: 14 and CDR3 ofSEQ ID NO: 16; (ii) a heavy chain variable region comprising a CDR1 ofSEQ ID NO: 20, a CDR2 of SEQ ID NO: 22 and CDR3 of SEQ ID NO: 24, and alight chain variable region comprising a CDR1 of SEQ ID NO: 28, a CDR2of SEQ ID NO: 30 and CDR3 of SEQ ID NO: 32; (iii) a heavy chain variableregion comprising a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 38 andCDR3 of SEQ ID NO: 40, and a light chain variable region comprising aCDR1 of SEQ ID NO: 44, a CDR2 of SEQ ID NO: 46 and CDR3 of SEQ ID NO:48; (iv) a heavy chain variable region comprising a CDR1 of SEQ ID NO:52, a CDR2 of SEQ ID NO: 54 and CDR3 of SEQ ID NO: 56, and a light chainvariable region comprising a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO:62 and CDR3 of SEQ ID NO: 64; (v) a heavy chain variable regioncomprising a CDR1 of SEQ ID NO: 68, a CDR2 of SEQ ID NO: 70 and CDR3 ofSEQ ID NO: 72, and a light chain variable region comprising a CDR1 ofSEQ ID NO: 76, a CDR2 of SEQ ID NO: 78 and CDR3 of SEQ ID NO: 80; (vi) aheavy chain variable region comprising a CDR1 of SEQ ID NO: 84, a CDR2of SEQ ID NO: 86 and CDR3 of SEQ ID NO: 88, and a light chain variableregion comprising a CDR1 of SEQ ID NO: 92, a CDR2 of SEQ ID NO: 94 andCDR3 of SEQ ID NO: 96; (vii) a heavy chain variable region comprising aCDR1 of SEQ ID NO: 100, a CDR2 of SEQ ID NO: 102 and CDR3 of SEQ ID NO:104, and a light chain variable region comprising a CDR1 of SEQ ID NO:108, a CDR2 of SEQ ID NO: 110 and CDR3 of SEQ ID NO: 112; (viii) a heavychain variable region comprising a CDR1 of SEQ ID NO: 116, a CDR2 of SEQID NO: 118 and CDR3 of SEQ ID NO: 120, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 124, a CDR2 of SEQ ID NO: 126 and CDR3of SEQ ID NO: 128; (ix) a heavy chain variable region comprising a CDR1of SEQ ID NO: 132, a CDR2 of SEQ ID NO: 134 and CDR3 of SEQ ID NO: 136,and a light chain variable region comprising a CDR1 of SEQ ID NO: 140, aCDR2 of SEQ ID NO: 142 and CDR3 of SEQ ID NO: 144; (x) a heavy chainvariable region comprising a CDR1 of SEQ ID NO: 148, a CDR2 of SEQ IDNO: 150 and CDR3 of SEQ ID NO: 152, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 156, a CDR2 of SEQ ID NO: 158 and CDR3of SEQ ID NO: 160; (xi) a heavy chain variable region comprising a CDR1of SEQ ID NO: 164, a CDR2 of SEQ ID NO: 166 and CDR3 of SEQ ID NO: 168,and a light chain variable region comprising a CDR1 of SEQ ID NO: 172, aCDR2 of SEQ ID NO: 174 and CDR3 of SEQ ID NO: 176; (xii) a heavy chainvariable region comprising a CDR1 of SEQ ID NO: 180, a CDR2 of SEQ IDNO: 182 and CDR3 of SEQ ID NO: 184, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 188, a CDR2 of SEQ ID NO: 190 and CDR3of SEQ ID NO: 192; (xiii) a heavy chain variable region comprising aCDR1 of SEQ ID NO: 196, a CDR2 of SEQ ID NO: 198 and CDR3 of SEQ ID NO:200, and a light chain variable region comprising a CDR1 of SEQ ID NO:204, a CDR2 of SEQ ID NO: 206 and CDR3 of SEQ ID NO: 208; (xix) a heavychain variable region comprising a CDR1 of SEQ ID NO: 210, a CDR2 of SEQID NO: 211 and CDR3 of SEQ ID NO: 212, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 214, a CDR2 of SEQ ID NO: 215 and CDR3of SEQ ID NO: 216; (xx) a heavy chain variable region comprising a CDR1of SEQ ID NO: 218, a CDR2 of SEQ ID NO: 219 and CDR3 of SEQ ID NO: 220,and a light chain variable region comprising a CDR1 of SEQ ID NO: 222, aCDR2 of SEQ ID NO: 223 and CDR3 of SEQ ID NO: 224; and/or (xxi) a heavychain variable region comprising a CDR1 of SEQ ID NO: 226, a CDR2 of SEQID NO: 227 and CDR3 of SEQ ID NO: 228, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 230, a CDR2 of SEQ ID NO: 231 and CDR3of SEQ ID NO:
 232. 22. The binding agent of claim 21, wherein thebinding agent has an affinity for the epitope of at least 10⁷ M⁻¹.
 23. Abinding agent comprising: (i) a heavy chain variable region comprising aCDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 6 and CDR3 of SEQ ID NO: 8,and a light chain variable region comprising a CDR1 of SEQ ID NO: 12, aCDR2 of SEQ ID NO: 14 and CDR3 of SEQ ID NO: 16; (ii) a heavy chainvariable region comprising a CDR1 of SEQ ID NO: 20, a CDR2 of SEQ ID NO:22 and CDR3 of SEQ ID NO: 24, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 30 and CDR3 ofSEQ ID NO: 32; (iii) a heavy chain variable region comprising a CDR1 ofSEQ ID NO: 36, a CDR2 of SEQ ID NO: 38 and CDR3 of SEQ ID NO: 40, and alight chain variable region comprising a CDR1 of SEQ ID NO: 44, a CDR2of SEQ ID NO: 46 and CDR3 of SEQ ID NO: 48; (iv) a heavy chain variableregion comprising a CDR1 of SEQ ID NO: 52, a CDR2 of SEQ ID NO: 54 andCDR3 of SEQ ID NO: 56, and a light chain variable region comprising aCDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 62 and CDR3 of SEQ ID NO:64; (v) a heavy chain variable region comprising a CDR1 of SEQ ID NO:68, a CDR2 of SEQ ID NO: 70 and CDR3 of SEQ ID NO: 72, and a light chainvariable region comprising a CDR1 of SEQ ID NO: 76, a CDR2 of SEQ ID NO:78 and CDR3 of SEQ ID NO: 80; (vi) a heavy chain variable regioncomprising a CDR1 of SEQ ID NO: 84, a CDR2 of SEQ ID NO: 86 and CDR3 ofSEQ ID NO: 88, and a light chain variable region comprising a CDR1 ofSEQ ID NO: 92, a CDR2 of SEQ ID NO: 94 and CDR3 of SEQ ID NO: 96; (vii)a heavy chain variable region comprising a CDR1 of SEQ ID NO: 100, aCDR2 of SEQ ID NO: 102 and CDR3 of SEQ ID NO: 104, and a light chainvariable region comprising a CDR1 of SEQ ID NO: 108, a CDR2 of SEQ IDNO: 110 and CDR3 of SEQ ID NO: 112; (viii) a heavy chain variable regioncomprising a CDR1 of SEQ ID NO: 116, a CDR2 of SEQ ID NO: 118 and CDR3of SEQ ID NO: 120, and a light chain variable region comprising a CDR1of SEQ ID NO: 124, a CDR2 of SEQ ID NO: 126 and CDR3 of SEQ ID NO: 128;(ix) a heavy chain variable region comprising a CDR1 of SEQ ID NO: 132,a CDR2 of SEQ ID NO: 134 and CDR3 of SEQ ID NO: 136, and a light chainvariable region comprising a CDR1 of SEQ ID NO: 140, a CDR2 of SEQ IDNO: 142 and CDR3 of SEQ ID NO: 144; (x) a heavy chain variable regioncomprising a CDR1 of SEQ ID NO: 148, a CDR2 of SEQ ID NO: 150 and CDR3of SEQ ID NO: 152, and a light chain variable region comprising a CDR1of SEQ ID NO: 156, a CDR2 of SEQ ID NO: 158 and CDR3 of SEQ ID NO: 160;(xi) a heavy chain variable region comprising a CDR1 of SEQ ID NO: 164,a CDR2 of SEQ ID NO: 166 and CDR3 of SEQ ID NO: 168, and a light chainvariable region comprising a CDR1 of SEQ ID NO: 172, a CDR2 of SEQ IDNO: 174 and CDR3 of SEQ ID NO: 176; (xii) a heavy chain variable regioncomprising a CDR1 of SEQ ID NO: 180, a CDR2 of SEQ ID NO: 182 and CDR3of SEQ ID NO: 184, and a light chain variable region comprising a CDR1of SEQ ID NO: 188, a CDR2 of SEQ ID NO: 190 and CDR3 of SEQ ID NO: 192;(xiii) a heavy chain variable region comprising a CDR1 of SEQ ID NO:196, a CDR2 of SEQ ID NO: 198 and CDR3 of SEQ ID NO: 200, and a lightchain variable region comprising a CDR1 of SEQ ID NO: 204, a CDR2 of SEQID NO: 206 and CDR3 of SEQ ID NO: 208; (xix) a heavy chain variableregion comprising a CDR1 of SEQ ID NO: 210, a CDR2 of SEQ ID NO: 211 andCDR3 of SEQ ID NO: 212, and a light chain variable region comprising aCDR1 of SEQ ID NO: 214, a CDR2 of SEQ ID NO: 215 and CDR3 of SEQ ID NO:216; (xx) a heavy chain variable region comprising a CDR1 of SEQ ID NO:218, a CDR2 of SEQ ID NO: 219 and CDR3 of SEQ ID NO: 220, and a lightchain variable region comprising a CDR1 of SEQ ID NO: 222, a CDR2 of SEQID NO: 223 and CDR3 of SEQ ID NO: 224; and/or (xxi) a heavy chainvariable region comprising a CDR1 of SEQ ID NO: 226, a CDR2 of SEQ IDNO: 227 and CDR3 of SEQ ID NO: 228, and a light chain variable regioncomprising a CDR1 of SEQ ID NO: 230, a CDR2 of SEQ ID NO: 231 and CDR3of SEQ ID NO:
 232. 24. The binding agent of claim 23, wherein saidbinding agent is not a natural antibody.